DNA Damage Signaling Pathways Research Articles

Organophosphorus Flame Retardant TCPP Induces Cellular Senescence in Normal Human Skin Keratinocytes: Implication for Skin Aging.

Tris (1-chloro-2-propyl) phosphate (TCPP) is one of the most frequently detected organophosphorus flames in the environment. Continuous daily exposure to TCPP may harm human skin. However, little is known about the adverse effects of TCPP on human skin. In this study, we first evaluated the detrimental effects and tried to uncover the underlying mechanisms of TCPP on human skin keratinocytes (HaCaT) after 24 h exposure. We found that TCPP caused a concentration-dependent decrease in HaCaT cell viability after exposure to 1.56-400 μg/mL for 24 h, with an IC50 of 275 μg/mL. TCPP also promoted the generation of intracellular reactive oxygen species (ROS) and triggered DNA damage, evidenced by an increase of phosphorylated histone H2A.X (γH2A.X) in the nucleus. Furthermore, the cell cycle was arrested at the G1 phase at 100 μg/mL by upregulation of the mRNA expression of p53 and p21 and downregulation of cyclin D1 and CDK4 expression. Additionally, both the senescence-associated-β-galactosidase activity and related proinflammatory cytokine IL-1β and IL-6 were elevated, indicating that TCPP exposure caused cellular senescence may be through the p53-dependent DNA damage signal pathway in HaCaT cells. Taken together, our data suggest that flame-retardant exposure may be a key precipitating factor for human skin aging.

Differential Gene Expression Profile in DNA Damage Signaling Pathways, in De Novo Acute Myeloid Leukemia Patients before Induction Chemotherapy

Introduction: Acute Myeloid Leukemia (AML) is a life-threatening myeloid malignancy with poor prognosis. Response to induction treatment determines long-term prognosis; however, mechanisms underlying response have not been thoroughly investigated. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also important for refractoriness to treatment. Aim: The aim of this study is to investigate the possible alterations in the gene expression profile of DNA damage signaling pathways in two leukemic cell lines following their exposure to chemotherapeutic agents, namely, idarubicin and cytarabine, and to verify the findings in AML patient samples at diagnosis before the onset of chemotherapy. Methods: Cell lines Kasumi-1 with the t(8;21) and MV4-11 (biphenotypic B-myelomonocytic leukemia) were exposed either to idarubicin (0.1μΜ) for 6h or cytarabine (1μΜ) for 48h. Dead cells were eliminated from drug-treated cells using an appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT2 Profiler, Qiagen) was performed in triplicate after RNA extraction from untreated, chemotherapy-treated and live (chemo resistant) cells following chemotherapy exposure. Human DNA damage signaling pathway-related gene expression and statistical analysis was performed through the RT2 Profiler PCR Array data analysis tool. Moreover, mononuclear cells were isolated from bone marrow of 11 lymphoma patients, used as controls, and from 64 AML patients at diagnosis who subsequently received chemotherapy with the 7+3 regimen. Response to first-line chemotherapy was recorded and patients were assigned in two groups: Responders (n=53 patients) and non-Responders (n=11 patients). Total RNA was isolated (RNeasy plus Mini Kit, Qiagen), quantified and cDNA was synthesized. Relative expression of genes of interest was quantified using SYBR® Greenbased real-time RT-PCR (QuantiTect® Primer Assay, Qiagen) based on the ddCt method (Housekeeping gene GADPH). All samples were obtained from patients after informed consent and treated in accordance with the Declaration of Helsinki. Statistical analysis was performed using R Statistical Software (version 4.2.1). Results: Five genes were found significantly [p < 0.05, Students t-test] up-regulated in live cells from either leukemic cell lines after treatment with both agents and had a FoldChange > 2. More specifically PPP1R15A, CDKN1A and GADD45G genes were up-regulated in both live cell lines, GADD45A in live MV4-11 cells and EXO1 in live Kasumi cells (Figure 1A). The aforementioned genes were selected and their expression levels were quantified in human bone marrow samples. CDKN1A and GADD45G were found down-regulated in Responders [p=0.032 and p=0.001 respectively; Welch t-test] while PPP1R15A was significantly up-regulated [over 8-fold change, p= 2.08E-09; Welch t-test] compared to controls. Likewise, GADD45G and PPP1R15A were also significantly down and up regulated respectively, in non-Responders [p=0.08 and p=1.51E-05, Welch t-test]. Interestingly, PPP1R15A presented a 1.7-fold overexpression trend in non-Responders compared to Responders [p=0.18, Welch t-test] (Figure 1B). Conclusion: The results of our study suggest a dysregulation of the DNA damage and repair pathways in AML. Upregulation of GADD45G is known to dramatically induce apoptosis, differentiation, and growth arrest while increasing sensitivity of AML cells to chemotherapeutic drugs (Guo, Dan et al., Blood 2021). CDKN1A is involved in p53/TP53 mediated inhibition of cellular proliferation in response to DNA damage. Both were down-regulated in AML patients and are likely to play an important role in the pathogenesis of AML. PPP1R15A participates in DNA damage response facilitating the recovery of cells from stress. The up-regulation of PPP1R15A both in live cells treated with Ara-C and Cytarabine, as well as the 1.7-fold overexpression trend in non-Responders compared to Responders, supports the notion that PPP1R15A could be associated with resistance to induction treatment, which may have significant prognostic and therapeutic implications. A greater sample of AML patients both responding and not responding to chemotherapy, would be essential to delineate the role of genes involved in DNA damage and repair pathways in resistance to induction treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting Sumoylation with Subasumstat (TAK-981), a Selective Inhibitor of SUMO-Activating Enzyme (SAE), Induces Mitochondrial Dysfunction in Preclinical Models of Non-Hodgkin Lymphoma (NHL)

SUMOylation is a post-translational modification which leads to covalent attachment of small ubiquitin-like modifiers (SUMO) proteins to lysine residues of a target protein. SUMOylation is essential for the regulation of genomic integrity, gene expression and intracellular signaling. Many tumors exhibit deregulation of SUMOylation pathway. Subasumstat, (TAK-981), a novel selective inhibitor of SUMO-activating enzyme (SAEi), has entered early phase II clinical trials. Here we studied SAEi in pre-clinical models of B-cell non-Hodgkin's lymphoma (NHL). TAK-981 was provided by Takeda Development Center Americas, Inc. (Lexington, MA). Diffuse large B-cell lymphoma (DLBCL) cell lines (OCI-LY3, VAL, U-2932, SU-DHL10), and primary mantle cell lymphoma (MCL) cells were assayed for survival, mitochondrial function, and metabolic phenotype. In vivo experiments were performed using DLBCL cell line xenografts and MCL PDX models. Treatment with TAK-981 restricted growth of DLBCL cell lines with an IC50~10 nM. SAEi led to rapid (within 2 h) protein desumoylation in a dose dependent manner, prominently in the ~90 kDa region. TAK-981 induced pronounced double strand DNA breaks in DLBCL cells (comet assay) and cell cycle arrest (propidium iodine). RNA-Seq and ATAC-Seq analysis of TAK-981-treated DLBCL cells revealed upregulation of the DNA damage, NF-κB, and OXPHOS signaling pathways. We noted that in addition to cytoplasmic proteins, mitochondrial proteins were heavily de-SUMOylated upon treatment of NHL cells with TAK-981. In vitro exposure to TAK-981 resulted in mitochondrial membrane depolarization, loss of mitochondrial integrity (by electron microscopy) and a change of mitochondrial dynamics as assessed by expression of mitochondrial proteins (eg, Drp1). Seahorse respirometry analysis revealed dramatic downmodulation of OXPHOS following SAEi. Furthermore, metabolomic profiling of DLBCL cells treated with TAK-981 showed downmodulation of TCA substrates, while glycolysis substrates remained unchanged. The mitochondrial disruption observed following TAK-981 treatment was accompanied by rapid accumulation of reactive oxygen species, a mechanism that has been previously shown to lead to DNA damage in cancer cells. Consistent with cell line data, exposure of primary MCL cells to TAK-981 in stromal conditions (CD40L- or BAFF expressing stroma) induced apoptosis and reduced OXPHOS. Next, we conducted genome-wide CRISPR-Cas9 loss-of-function library screens and identified that loss of genes in the NFκB, TP53, DNA damage and centromere/telomere gene pathways contributed to resistance to SAEi in DLBCL cells. By means of genetic knockout we demonstrated that TP53 and its transcriptional target BAX contributed to TAK-981-induced G2 arrest and apoptosis, further implicating mitochondrial dysfunction in sensitivity to SAEi. For in vivo experiments, we used DLBCL xenografts. OCI-LY3 and U-2932 cells were inoculated subcutaneously in NSG mice. Once tumors reached 100 mm3, mice received 10 twice-weekly IV doses of 7.5 mg/kg TAK-981 (over 5 weeks), leading to resolution of flank tumors and improved survival compared with control. Finally, we used an MCL PDX model where MCL cells were inoculated intravenously. Once cells became detectable in the blood, mice were treated as above. Treatment with TAK-981 resulted in delayed tumor expansion in blood and spleen and extended animal survival. TAK-981-exposed splenocytes exhibited a reduction of maximal respiration by Seahorse analysis. Conclusions. Pharmacologic SAEi with TAK-981 demonstrated pre-clinical activity in NHL models in vitro and in vivo, accompanied by DNA damage, mitochondrial dysfunction, and metabolic reprogramming.

The Interplay between the Cellular Response to DNA Double-Strand Breaks and Estrogen.

Cancer development is often connected to impaired DNA repair and DNA damage signaling pathways. The presence of DNA damage in cells activates DNA damage response, which is a complex cellular signaling network that includes DNA repair, activation of the cell cycle checkpoints, cellular senescence, and apoptosis. DNA double-strand breaks (DSBs) are toxic lesions that are mainly repaired by the non-homologous end joining and homologous recombination repair (HRR) pathways. Estrogen-dependent cancers, like breast and ovarian cancers, are frequently associated with mutations in genes that play a role in HRR. The female sex hormone estrogen binds and activates the estrogen receptors (ERs), ERα, ERβ and G-protein-coupled ER 1 (GPER1). ERα drives proliferation, while ERβ inhibits cell growth. Estrogen regulates the transcription, stability and activity of numerus DDR factors and DDR factors in turn modulate ERα expression, stability and transcriptional activity. Additionally, estrogen stimulates DSB formation in cells as part of its metabolism and proliferative effect. In this review, we will present an overview on the crosstalk between estrogen and the cellular response to DSBs. We will discuss how estrogen regulates DSB signaling and repair, and how DDR factors modulate the expression, stability and activity of estrogen. We will also discuss how the regulation of HRR genes by estrogen promotes the development of estrogen-dependent cancers.

Blockades of effector T cell senescence and exhaustion synergistically enhance antitumor immunity and immunotherapy

BackgroundCurrent immunotherapies still have limited successful rates among cancers. It is now recognized that T cell functional state in the tumor microenvironment (TME) is a key determinant for effective antitumor...

Adrenal histological and functional changes after hepatic encephalopathy: From mice model to an integrative bioinformatics analysis

Hepatic encephalopathy (HE), which is caused by neurotoxin agents in the liver, is a complicated condition with a variety of neurological manifestations. Recently, endocrine alterations have been more paid attention to for neurological severity in the course of HE, e.g. adrenal gland. To identify the role of adrenal gland in the context of HE, we evaluated the functional changes of adrenal gland (i.e., plasma corticosterone concentrations and histopathological changes) in mice model of HE. To dig deep into the molecular and genetic underpinnings, a comprehensive enrichment analysis for shared genes between HE and adrenal insufficiency (AI) was also performed. Our results showed a significant reduction in the level of plasma corticosterone and severe cellular necrosis in zona fasciculate of adrenal cortex, possibly indicating adrenal insufficiency. Enrichment analysis indicated four common genes, besides predicted five novel genes and some significant MicroRNAs (miRNAs) and transcription factors for both HE and AI. Couples with, several biological processes, such as DNA damage, inflammatory responses, glycolytic processes, and insulin receptor signaling pathway were predicted in both HE and AI. To sum up, data from experimental tests and bioinformatics analyses suggest that AI play an important role in the pathogenesis and progression of HE.

Circadian Clock Genes Act as Diagnostic and Prognostic Biomarkers of Glioma: Clinic Implications for Chronotherapy.

Gliomas are the most common primary intracranial tumors and closely related to circadian clock. Due to the high mortality and morbidity of gliomas, exploring novel diagnostic and early prognostic markers is necessary. Circadian clock genes (CCGs) play important roles in regulating the daily oscillation of biological processes and the development of tumor. Therefore, we explored the influences that the oscillations of circadian clock genes (CCGs) on diagnosis and prognosis of gliomas using bioinformatics. In this work, we systematically analyzed the rhythmic expression of CCGs in brain and found that some CCGs had strong rhythmic expression; the expression levels were significantly different between day and night. Four CCGs (ARNTL, NPAS2, CRY2, and DBP) with rhythmic expression were not only identified as differentially expressed genes but also had significant independent prognostic ability in the overall survival of glioma patients and were highly correlated with glioma prognosis in COX analysis. Besides, we found that CCG-based predictive model demonstrated higher predictive accuracy than that of the traditional grade-based model; this new prediction model can greatly improve the accuracy of glioma prognosis. Importantly, based on the four CCGs' circadian oscillations, we revealed that patients sampled at night had higher predictive ability. This may help detect glioma as early as possible, leading to early cancer intervention. In addition, we explored the mechanism of CCGs affecting the prognosis of glioma. CCGs regulated the cell cycle, DNA damage, Wnt, mTOR, and MAPK signaling pathways. In addition, it also affects prognosis through gene coexpression and immune infiltration. Importantly, ARNTL can rhythmically modulated the cellular sensitivity to clinic drugs, temozolomide. The optimal point of temozolomide administration should be when ARNTL expression is highest, that is, the effect is better at night. In summary, our study provided a basis for optimizing clinical dosing regimens and chronotherapy for glioma. The four key CCGs can serve as potential diagnostic and prognostic biomarkers for glioma patients, and ARNTL also has obvious advantages in the direction of glioma chronotherapy.

Research Progress of DUB Enzyme in Hepatocellular Carcinoma.

According to GLOBOCAN 2021 cancer incidence and mortality statistics compiled by the International Agency for Research on Cancer, hepatocellular carcinoma (HCC) is the most common malignancy in the human liver and one of the leading causes of cancer death worldwide. Although there have been great advances in the treatment of HCC, such as regofenib, sorafenib, and lomvatinib, which have been developed and approved for the clinical treatment of advanced or metastatic HCC. However, they only prolong survival by a few months, and patients with advanced liver cancer are susceptible to tumor invasion metastasis and drug resistance. Ubiquitination modification is a type of post-translational modification of proteins. It can affect the physiological activity of cells by regulating the localization, stability and activity of proteins, such as: gene transcription, DNA damage signaling and other pathways. The reversible process of ubiquitination is called de-ubiquitination: it is the process of re-releasing ubiquitinated substrates with the participation of de-ubiquitinases (DUBs) and other active substances. There is growing evidence that many dysregulations of DUBs are associated with tumorigenesis. Although dysregulation of deuquitinase function is often found in HCC and other cancers, The mechanisms of action of many DUBs in HCC have not been elucidated. In this review, we focused on several deubiquitinases (DUBs) associated with hepatocellular carcinoma, including their structure, function, and relationship to hepatocellular carcinoma. hepatocellular carcinoma was highlighted, as well as the latest research reports. Among them, we focus on the USP family and OTU family which are more studied in the HCC. In addition, we discussed the prospects and significance of targeting DUBs as a new strategy for the treatment of hepatocellular carcinoma. It also briefly summarizes the research progress of some DUB-related small molecule inhibitors and their clinical application significance as a treatment for HCC in the future.

PB1787: GADD45A IS OVER-EXPRESSED IN THE BONE MARROW OF PATIENTS WITH DE NOVO ACUTE MYELOID LEUKEMIA REFRACTORY TO INDUCTION CHEMOTHERAPY

Background: Acute myeloid leukemia (AML) is a life-threatening malignant myeloid disorder with poor prognosis. Response to induction treatment determines long-term prognosis; however, mechanisms underlying response have not been thoroughly investigated. DNA damage and repair mechanisms influence not only the genetic predisposition to leukemia but are also very important for refractoriness to treatment. Aims: The aim of this study was to investigate the possible alterations in the gene expression profile of DNA damage signaling pathways in two leukemic cell lines following their exposure to chemotherapeutic agents, namely, idarubicin and cytarabine, and verify the findings in AML patient samples at diagnosis before the onset of chemotherapy. Methods: Cell lines Kasumi-1 with the t(8;21) and MV4-11 (biphenotypic B-myelomonocytic leukemia) were exposed either to idarubicin (0.1μΜ) for 6h or cytarabine (1μΜ) for 48h. Dead cells were eliminated from drug-treated cells using an appropriate commercial kit. Gene expression profiling through PCR arrays analysis (RT2 Profiler, Qiagen) was performed in triplicate after RNA extraction from untreated, chemotherapy-treated and live cells following chemotherapy exposure. Human DNA damage signaling pathway-related gene expression was evaluated and analyzed through the RT2 Profiler PCR Array data analysis tool. Moreover, mononuclear cells were isolated from bone marrow of lymphoma patients, used as controls, and from AML patients at diagnosis who subsequently received chemotherapy with the 7 + 3 regiment. Response to first-line chemotherapy was recorded. Total RNA was isolated (RNeasy plus Mini Kit, Qiagen) and quantified and cDNA was synthesized. Relative expression of genes of interest was quantified using SYBR® Green-based real-time RT-PCR (QuantiTect® Primer Assay, Qiagen). Statistics were performed using linear regression. Results: The following genes were over two-fold up-regulated in live cells from leukemic cell lines after treatment with either agent: PPP1R15A and CDKN1A in both cell lines, GADD45A and GADD45G in live MV4-11 cells and EXO1 in live Kasumi cells. These genes were selected for relative quantification; 25 control samples and 38 AML samples (28 responders and 10 non-responders to treatment) were evaluated. GADD45A in AML compared to controls was found 2-fold up-regulated in treatment responders (ns) and 9-fold up-regulated in non-responders [p=0.01]. In addition, GADD45A was overexpressed in non-responders compared to responders [p=0.02]. PPP1R15A in AML compared to controls was 12-fold up-regulated in responders [p=0.001] and 16-fold up-regulated in non-responders [p=0.001]. (Figure 1) Image:Summary/Conclusion: GADD45A controls the balance between apoptosis and autophagy via the p38 and JNK/MAPK pathways and PPP1R15A participates in growth arrest and apoptosis in response to DNA damage by attenuating the translational elongation of key transcription factors. Both genes are likely to play an important role in the pathogenesis of AML. Furthermore, GADD45A overexpression appears to be related with resistance to induction treatment, which may have significant prognostic and therapeutic implications.

Interplay between H3K36me3, methyltransferase SETD2, and mismatch recognition protein MutSα facilitates processing of oxidative DNA damage in human cells

Oxidative DNA damage contributes to aging and the pathogenesis of numerous human diseases including cancer. 8-hydroxyguanine (8-oxoG) is the major product of oxidative DNA lesions. Although OGG1-mediated base excision repair is the primary mechanism for 8-oxoG removal, DNA mismatch repair has also been implicated in processing oxidative DNA damage. However, the mechanism of the latter is not fully understood. Here, we treated human cells defective in various 8-oxoG repair factors with H2O2 and performed biochemical, live cell imaging, and chromatin immunoprecipitation sequencing analyses to determine their response to the treatment. We show that the mismatch repair processing of oxidative DNA damage involves cohesive interactions between mismatch recognition protein MutSα, histone mark H3K36me3, and H3K36 trimethyltransferase SETD2, which activates the ATM DNA damage signaling pathway. We found that cells depleted of MutSα or SETD2 accumulate 8-oxoG adducts and fail to trigger H2O2-induced ATM activation. Furthermore, we show that SETD2 physically interacts with both MutSα and ATM, which suggests a role for SETD2 in transducing DNA damage signals from lesion-bound MutSα to ATM. Consistently, MutSα and SETD2 are highly coenriched at oxidative damage sites. The data presented here support a model wherein MutSα, SETD2, ATM, and H3K36me3 constitute a positive feedback loop to help cells cope with oxidative DNA damage.

The Combination of a Novel GLUT1 Inhibitor and Cisplatin Synergistically Inhibits Breast Cancer Cell Growth By Enhancing the DNA Damaging Effect and Modulating the Akt/mTOR and MAPK Signaling Pathways.

Breast cancer is the most prevalent cancer and the second leading cause of cancer death in women. Cisplatin is a commonly used chemotherapeutic drug for breast cancer treatment. Owing to serious side effects, the combination of cisplatin with other drugs is an effective strategy to simultaneously reduce side effects and increase the anticancer efficacy. GLUT1 is an emerging target for cancer treatment since cancer cells usually consume more glucose, a phenomenon called the Warburg effect. In this study, we found that the combination of cisplatin and a novel GLUT1 inhibitor #43 identified from our previous high-throughput screening exerted a synergistic anticancer effect in MCF-7 and MDA-MB-231 breast cancer cells. Mechanism studies in MCF-7 cells revealed that combination of cisplatin and #43 significantly induced apoptosis, intracellular reactive oxygen species, and loss of mitochondrial membrane potential. Furthermore, #43 enhanced the DNA damaging effect of cisplatin. Akt/mTOR downstream signaling and the ERK signaling pathway usually involved in cell growth and survival were inhibited by the combination treatment. On the other hand, phosphorylation of p38 and JNK, which may be associated with apoptosis, was induced by the combination treatment. Altogether, our data indicate that oxidative stress, DNA damage, the Akt/mTOR and MAPK signaling pathways, and apoptosis may be involved in the synergism of cisplatin and #43 in breast cancer cells.

Phosphorylation regulates E3 ligase activity of Mdm2

The Mdm2 oncogene is associated with the negative regulation of the tumor suppressor p53 in an autoregulatory feedback loop in response to DNA damage. It has been assumed that Mdm2 functions in this pathway as an E3 ubiquitin ligase and is Mdm2’s principal function under all conditions. However, the E3 ligase activity can function either as ubiquitin or nedd8 E3 ligase. This activity is dependent on specific tyrosine kinases that are activated in response to DNA damage signaling or growth factor signaling pathways. The E3 ligase activity can have a distinct effect on the substrate by either stabilizing or signaling destruction. In the context of growth signaling pathways, we show that Src phosphorylation of Mdm2 recruits Ubc12 (nedd8 E2 enzyme). This results in the neddylation of the tumor suppressors pVHL and p53. The neddylation of pVHL prevents the association with p53, thereby attenuating the induction of anti‐angiogenic genes. The phosphorylation of Mdm2 by Src is in the Zinc domain, and we found that this domain is a key regulatory domain for the recruitment of E2 enzymes. These findings suggest that the role of Mdm2 is more complex than just functioning as an E3 ubiquitin ligase to signal for the destruction of p53.

The diaryl-imidazopyridazine anti-plasmodial compound, MMV652103, exhibits anti-breast cancer activity

Breast cancer is the most common malignancy in women worldwide and it remains a global health burden, in part, due to poor response and tolerance to current therapeutics. Drug repurposing, which seeks to identify new indications for existing and investigational drugs, has become an exciting strategy to address these challenges. Here we describe the anti-breast cancer activity of a diaryl-imidazopyridazine compound, MMV652103, which was previously identified for its anti-plasmodial activity. We demonstrate that MMV652103 potently inhibits the oncogenic PI4KB and PIK3C2G lipid kinases, is selectively cytotoxic to MCF7 and T47D estrogen receptor positive breast cancer cells and inhibits their ability to survive and migrate. The underlying mechanisms involved included the induction of reactive oxygen species and activation of the DNA damage and p38 MAPK stress signaling pathways. This was associated with a G1 cell cycle arrest and an increase in levels of the cyclin-dependent kinase inhibitor p21 and activation of apoptotic and autophagic cell death pathways. Lastly, MMV652103 significantly reduced the weight and metastases of MCF7 induced tumors in an in vivo chick embryo model and displayed a favorable safety profile. These findings position MMV652103 as a promising chemotherapeutic in the treatment of oestrogen receptor positive breast cancers.

DNA repair defects in cancer and therapeutic opportunities.

DNA repair and DNA damage signaling pathways are critical for the maintenance of genomic stability. Defects of DNA repair and damage signaling contribute to tumorigenesis, but also render cancer cells vulnerable to DNA damage and reliant on remaining repair and signaling activities. Here, we review the major classes of DNA repair and damage signaling defects in cancer, the genomic instability that they give rise to, and therapeutic strategies to exploit the resulting vulnerabilities. Furthermore, we discuss the impacts of DNA repair defects on both targeted therapy and immunotherapy, and highlight emerging principles for targeting DNA repair defects in cancer therapy.

Isoalantolactone Enhances the Antitumor Activity of Doxorubicin by Inducing Reactive Oxygen Species and DNA Damage.

Colon cancer is one of the most common cancer in the world. Doxorubicin (DOX) is a classical anti-tumor drug which widely used in treatment of cancers, however, high toxicity limited its further clinical application. Thus, it is urgent to find new drugs with low toxicity and high efficiency to treat colon cancer. Isoalantolactone (IATL), an isomeric sesquiterpene lactone isolated from the plant of inula helenium, has been reported to have anti-cancer activity against a variety of cancer cells. However, the function of IATL in colon cancer remains unclear. Here, we demonstrated that IATL inhibited colon cancer cell growth by increasing cellular reactive oxygen species (ROS) production. Further study showed that ROS accumulation contributed to DNA damage and JNK signaling pathway activation. In addition, we found that IATL markedly enhanced DOX-induced cell cytotoxicity in colon cancer cells. IATL in combination with DOX significantly increased the ROS production, induced DNA damage and activated JNK signaling pathway. Taken together, our data suggested that combined treatment with IATL and DOX may serve as a potential therapeutics for colon cancer.

Generation of Monoubiquitin and K63-Linked Polyubiquitin Chains for Protein Interaction Studies.

Ubiquitylation is a posttranslational modification that utilizes protein-protein binding interactions to regulate cellular processes. In ubiquitin signaling, a vast array of mono- and polyubiquitin modifications to substrate proteins are recognized by a diverse group of ubiquitin-binding proteins. Identifying ubiquitin-binding proteins and characterizing their binding properties is necessary for understanding the structural basis of ubiquitin signaling. This chapter provides a means of studying ubiquitin-binding interactions in vitro by describing how to generate monoubiquitin and K63-linked polyubiquitin chains and perform pull-down assays with ubiquitin-binding proteins, which is of particular relevance for DNA damage and other signaling pathways.

Attention-based Long Short Term Memory Model for DNA Damage Prediction in Mammalian Cells

The understanding of DNA damage intensity – concentration-level is critical for biological and biomedical research, such as cellular homeostasis, tumor suppression, immunity, and gametogenesis. Therefore, recognizing and quantifying DNA damage intensity levels is a substantial issue, which requires further robust and effective approaches. DNA damage has several intensity levels. These levels of DNA damage in malignant cells and in other unhealthy cells are significant in the assessment of lesion stages located in normal cells. There is a need to get more insight from the available biological data to predict, explore and classify DNA damage intensity levels. Herein, the development process relied on the available biological dataset related to DNA damage signaling pathways, which plays a crucial role in DNA damage in the mammalian cell system. The biological dataset that was used in the proposed model consists of 15000 records intensity – concentration-level for a set of five proteins which regulate DNA damage. This research paper proposes an innovative deep learning model, which consists of an attention-based long short term-memory (AT-LSTM) model for DNA damage multi class predictions. The proposed model splits the prediction procedure into dual stages. For the first stage, we adopt the related feature sequences which are inserted as input to the LSTM neural network. In the next stage, the attention feature is applied efficiently to adopt the related feature sequences which are inserted as input to the softmax layer for prediction in the following frame. Our developed framework not only solves the long-term dependence problem of prediction effectively, but also enhances the interpretability of the prediction methods that was established on the neural network. We conducted a novel proposed model on big and complex biological datasets to perform prediction and multi classification tasks. Indeed, the (AT-LSTM) model has the ability to predict and classify the DNA damage in several classes: No-Damage, Low-damage, Medium-damage, High-damage, and Excess-damage. The experimental results show that our framework for DNA damage intensity level can be considered as state of the art for the biological DNA damage prediction domain.

Simultaneous loss of TSC1 and DEPDC5 in skeletal and cardiac muscles produces early-onset myopathy and cardiac dysfunction associated with oxidative damage and SQSTM1/p62 accumulation

ABSTRACT By promoting anabolism, MTORC1 is critical for muscle growth and maintenance. However, genetic MTORC1 upregulation promotes muscle aging and produces age-associated myopathy. Whether MTORC1 activation is sufficient to produce myopathy or indirectly promotes it by accelerating tissue aging is elusive. Here we examined the effects of muscular MTORC1 hyperactivation, produced by simultaneous depletion of TSC1 and DEPDC5 (CKM-TD). CKM-TD mice produced myopathy, associated with loss of skeletal muscle mass and force, as well as cardiac failure and bradypnea. These pathologies were manifested at eight weeks of age, leading to a highly penetrant fatality at around twelve weeks of age. Transcriptome analysis indicated that genes mediating proteasomal and macroautophagic/autophagic pathways were highly upregulated in CKM-TD skeletal muscle, in addition to inflammation, oxidative stress, and DNA damage signaling pathways. In CKM-TD muscle, autophagosome levels were increased, and the AMPK and ULK1 pathways were activated; in addition, autophagy induction was not completely blocked in CKM-TD myotubes. Despite the upregulation of autolysosomal markers, CKM-TD myofibers exhibited accumulation of autophagy substrates, such as SQSTM1/p62 and ubiquitinated proteins, suggesting that the autophagic activities were insufficient. Administration of a superoxide scavenger, tempol, normalized most of these molecular pathologies and subsequently restored muscle histology and force generation. However, CKM-TD autophagy alterations were not normalized by rapamycin or tempol, suggesting that they may involve non-canonical targets other than MTORC1. These results collectively indicate that the concomitant muscle deficiency of TSC1 and DEPDC5 can produce early-onset myopathy through accumulation of oxidative stress, which dysregulates myocellular homeostasis. Abbreviations: AMPK: AMP-activated protein kinase; CKM: creatine kinase, M-type; COX: cytochrome oxidase; DEPDC5: DEP domain containing 5, GATOR1 subcomplex subunit; DHE: dihydroethidium; EDL: extensor digitorum longus; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; GAP: GTPase-activating protein; GTN: gastrocnemius; MTORC1: mechanistic target of rapamycin kinase complex 1; PLA: plantaris; QUAD: quadriceps; RPS6KB/S6K: ribosomal protein S6 kinase beta; SDH: succinate dehydrogenase; SOL: soleus; SQSTM1: sequestosome 1; TA: tibialis anterior; TSC1: TSC complex subunit 1; ULK1: unc-51 like autophagy activating kinase 1

Analysis of the mechanism underlying the effects of cyclophosphamide against triple-negative breast cancer by an integrative bioinformatics approach

Patients with Triple-Negative Breast Cancer (TNBC), a highly heterogeneous and invasive subtype of breast cancer, do not benefit from hormonal therapy or trastuzumab; therefore, chemotherapy is considered the only option. We explored the effect of the chemotherapeutic drug cyclophosphamide (Cytoxan; Cy) on TNBC by an integrated bioinformatics approach. Methods: Pharm Mapper, Gene Cards, and Swiss Target Prediction were used to identify potential targets of Cy. Differentially expressed genes (DEGs) in TNBC were screened out from four GEO datasets. Common genes were further evaluated by a protein-protein interaction network analysis, core gene identification, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, docking assays, and survival and immune cell infiltration analyses. Results: We collected information on 1638 drug targets and 494 DEGs in TNBC, including 267 up-regulated and 227 down-regulated genes. In total, 68 overlapping genes were identified as common targets. Ten core genes were identified in network analysis; GO and KEGG analyses revealed enrichment for DNA damage and many signaling pathways. Four core gene targets were verified by molecular docking. Kaplan–Meier analysis revealed that two core genes were significantly related to an adverse overall survival; furthermore, immune infiltration analysis suggested that Cy affects the microenvironment. Conclusions: Our integrative bioinformatics approach revealed that the anti-TNBC effect of Cy was mediated by DNA damage-related genes and many pathways. These findings provide a basis for further functional studies aimed at improving outcomes in TNBC.

DDRE-40. BIOINFORMATICS ANALYSIS OF GRADE I AND III MENINGIOMA GENE EXPRESSION SUGGESTS MMP28 AS A POSSIBLE POSITIVE PROGNOSTIC INDICATOR

Abstract Meningiomas are the most common primary brain tumor. Grade I meningiomas are slow growing with a good prognosis following surgical resection. High grade meningiomas (II and III) are more aggressive with greater rates of recurrence and poor prognosis despite surgical resection and radiotherapy. This study sought to identify key pathways and genes that contribute to the molecular mechanisms that separate these distinct meningioma phenotypes. FFPE specimens were evaluated and graded by a pathologist. Total RNA was extracted from tissue curls and RNA samples were measured with the Illumina DASL whole genome gene expression assay (n = 29,374 probes). A log2 transformation was applied to all of the data and quantile normalization using 21 samples. A t-test of the log2 expression of the 10 grade III samples and 11 grade I samples resulted in 1505 differentially expressed genes. The GeneCard Suite’s GeneAnalytics tool was used to perform pathway enrichment analysis. Similar to other comparable data sets, DNA Damage, Cell Cycle, EGF/EGFR Signaling, TGF-Beta Signaling, and PI3K-AKT Signaling pathways were among the 20 enriched pathways. Notably, matrix metalloprotease 28 (MMP28, also known as epilysin) was down regulated in grade III meningiomas compared to grade I (III/I expression ratio 0.315; P=0.015). MMP28 is expressed in numerous tissues including the nervous system during development and regeneration. In cancer, MMP28 is thought to enhance epithelial to mesenchymal transition by altering the TGF-B signaling pathways. MMP28 is an indicator of poor prognosis for pancreatic, lung, gastric and hepatic cancers as well as glioblastoma. However, MMP28 has also been reported as down regulated during malignant transformation in colon cancer. Our data suggest that MMP28 may have an alternative role in meningioma and serve as a positive prognostic indicator.