Induction Of DNA Damage Response Research Articles

Abstract 1712: Estrogen receptor beta signaling sensitizes glioblastoma cells to chemo and radiation therapy

Abstract Current treatment options for glioblastoma (GBM) are poor, the mortality rates are very high and therapy resistance is a major clinical problem. Standard treatment consists of surgical resection, external beam radiation therapy, adjuvant chemotherapy with temozolomide (TMZ), and tumor treating fields. Nonetheless, despite a heavy investment in therapy, all patients eventually succumb to their disease. Recent studies suggest that estrogen receptor beta (ERβ) may function as a tumor suppressor in GBM. However, the mechanism(s) by which ERβ contributes to chemo- and radiation therapy response remains unknown. The objective of this study is to examine whether ERβ sensitizes GBM to chemo- and radiation therapy and to understand the mechanistic insights of ERβ mediated tumor suppression in GBM. Methods: To study the functions of endogenous ERβ in GBM cells, we have utilized multiple ERβ overexpressing GBM model (GBM- ERβ) cells using lentiviral transduction. As a second model we have also generated ERβ knockout (ERβKO) cells using CRISPR/Cas9 system, and as a third model we used lentiviral-ERβshRNA transfected primary GBM cells (ERβKD). We then examined the effect of TMZ and radiation on the expression of ERβ using qRT-PCR. The effect of TMZ and radiation on ERβ overexpression and knockout models was examined using MTT cell viability assays. Mechanistic studies were conducted using RNA-seq, HR reporter gene assays, confocal microscopy, western blot, and qRT-PCR analysis. The in vivo role of ERβ on chemo sensitivity of TMZ was studied using orthotopic models of GBM and mouse survival was determined using Kaplan-Meier survival curves. Results: Cell viability and survival assays using multiple established and primary GBM cells demonstrated that ERβ sensitizes GBM cells to DNA damaging agents including TMZ and radiation therapy. qRT-PCR analysis demonstrated that ERβ expression was decreased following chemo- and radiation treatment. Combination analysis of RNA-seq studies using ERβ overexpression, and ERKO models, revealed an alteration in the number of genes involved in DNA recombination and repair, ATM signaling, and cell cycle check point control. Mechanistic studies showed that ERβ plays a significant role in homologous recombination (HR) mediated repair; and ERβ reduced expression and activation of ATM upon DNA damage. Generation of optimal γH2AX foci following TMZ treatment is dependent on the status of ERβ. More importantly, GBM cells expressing ERβ had increased survival when compared to control GBM cells in orthotopic GBM models. ERβ overexpression further enhanced the survival of mice to TMZ therapy in both TMZ sensitive and TMZ resistant GBM models. Conclusion: Our results provided evidence that ERβ is required for optimal chemo- and radiation- induced DNA damage response in GBM cells. Citation Format: Uday P. Pratap, Gangadhara Reddy Sareddy, Mengxing Li, Yiliao Luo, Mei Zhou, Suryavathi Viswanadhapalli, Rajeshwar Rao Tekmal, Andrew Brenner, Ratna K. Vadlamudi. Estrogen receptor beta signaling sensitizes glioblastoma cells to chemo and radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1712.

Abstract 2537: Targeting chromosomal passenger complex by disruption of INCENP function inhibits tumor progression in neuroblastoma

Abstract Chromosomal passenger complex (CPC), which is composed of Aurora B and Survivin, INCENP and Borealin, regulates crucial mitotic events including chromosome alignment, segregation and cytokinesis during the mitosis. Disruption of CPC function by Aurora B and Survivin inhibition has been demonstrated to be effective at killing tumor cells and show promising results in many different cancer types including neuroblastoma (NB). However, chemical inhibition of either Aurora B or Survivin is unable to target CPC specifically due to off-target effects of Aurora B inhibitors on the other kinases and antiapoptotic activities of Survivin independent of this complex. In a chromatin-focused siRNA screen, we found that NB cells were particularly vulnerable to the silencing of INCENP, a gene encoding a key scaffolding and regulatory component of the CPC. In this study, we found that INCENP was highly expressed in NB cells and its expression levels were decreased upon Retinoic Acid (RA)-induced NB cell differentiation. Genetic silencing of INCENP reduced the growth of both MYCN single copy and MYCN amplified NB cell lines in vitro and led to significant decreases in NB xenograft growth and increases in murine survival in vivo. Elevated levels of INCENP were significantly associated with poor prognosis in primary NB tumors whereas low INCENP expression levels were predictive of better outcomes. Mechanistically, we found that INCENP depletion suppressed NB cell growth by inducing massive polyploidization, mitotic arrest, senescence and cell death (apoptosis). We also observed that in the majority of NB cell lines tested in vitro, cell death represented the primary cell fate after INCENP silencing due to a strong induction of DNA damage response and activation of the p53-p21 axis. Therefore, targeting INCENP phenocopies treatment with Aurora B inhibitor, providing a novel strategy to disrupt the activity of CPC and inhibit tumor progression in NB. Citation Format: Ming Sun, Veronica Veschi, Norris Lam, Sukriti Bagchi, Man Xu, Arnulfo Mendoza, Zhihui Liu, Carol J. Thiele. Targeting chromosomal passenger complex by disruption of INCENP function inhibits tumor progression in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2537.

VE-822 mediated inhibition of ATR signaling sensitizes chondrosarcoma to cisplatin via reversion of the DNA damage response.

IntroductionCisplatin has been reported to elicit the DNA damage response (DDR) via activation of the ATR-Chk1 pathway, which in turn contributes to the induction of cisplatin resistance. Inhibition of ATR-Chk1 signaling reverses cisplatin resistance in some cancers. However, the influence of inhibiting ATR-Chk1 signaling on cisplatin resistance in chondrosarcoma cancer has not been reported.Materials and methodsWe compared the expression levels of ATR kinases in human nasopharyngeal carcinoma, choriocarcinoma and chondrosarcoma cell lines. We inhibited ATR kinase function with VE-822, a selective ATR inhibitor, and suppressed ATR kinase expression with shRNA. Western blotting, the CCK-8 assay, cell cycle distribution assay and apoptosis analysis were used to study the influence of inhibiting ATR-Chk1 signaling on reversing cisplatin resistance in chondrosarcoma cell lines.ResultsWe found that chondrosarcoma cells expressed very low basal levels of phosphorylated ATR, but cisplatin treatment induced the activation of ATR-Chk1 signaling in a dose- and time-dependent manner, suggesting the induction of DDR. As expected, ATR inhibition with VE-822 reversed cisplatin-induced DDR and enhanced cisplatin-induced activation of H2AX, which is an important marker of DNA damage. Meanwhile, ATR inhibition by RNA interference also reversed DDR and promoted DNA damage. Furthermore, both pharmacological and molecular inhibition of ATR accelerated cisplatin-induced inhibition of cell proliferation and cell death.ConclusionOur results suggested that inhibiting ATR activation promoted cisplatin-induced cell death via reversion of DDR, and VE-822 may be a valuable strategy for the prevention of cisplatin resistance in chondrosarcoma.

Ribosomal DNA as DAMPs Signal for MCF7 Cancer Cells

Introduction: The cell free ribosomal DNA (cf-rDNA) is accrued in the total pool of cell free DNA (cfDNA) in some non-cancer diseases and demonstrates DAMPs characteristics. The major research questions: (1) How does cell free rDNA content change in breast cancer; (2) What type of response in the MCF7 breast cancer cells is caused by cf-rDNA; and (3) What type of DNA sensors (TLR9 or AIM2) is stimulated in MCF7 in response to the action of cf-rDNA?Materials and Methods: CfDNA and gDNA were isolated from the blood plasma and the cells derived from 38 breast cancer patients and 20 healthy female controls. The rDNA content in DNA was determined using non-radioactive quantitative hybridization. In order to explore the rDNA influence on MCF7 breast cancer cells, the model constructs (GC-DNAs) were applied: pBR322-rDNA plasmid (rDNA inset 5836 bp long) and pBR322 vector. ROS generation, DNA damage, cell cycle, expression of TLR9, AIM2, NF-kB, STAT3, and RNA for 44 genes affecting the cancer cell viability were evaluated. The methods used: RT-qPCR, fluorescent microscopy, immunoassay, flow cytometry, and siRNA technology.Results: The ratio R = cf-rDNA/g-rDNA for the cases was higher than for the controls (median 3.4 vs. 0.8, p < 10−8). In MCF7, GC-DNAs induce a ROS burst, DNA damage response, and augmentation of NF-kB and STAT3 activity. The number of the apoptotic cells decreases, while the number of cells with an instable genome (G2/M– arrest, micronuclei) increase. Expression of anti-apoptotic genes (BCL2, BCL2A1, BCL2L1, BIRC3, MDM2) is elevated, while expression of pro-apoptotic genes (BAX, BID, BAD, PMAIP1, BBC3) is lowered. The cells response for pBR322-rDNA is much more intense and develops much faster, than response for pBR322, and is realized through activation of TLR9- MyD88 - NF-kB- signaling. This difference in response speed is owing to the heightened oxidability of pBR322-rDNA and better ability to penetrate the cell. Induction of TLR9 expression in MCF7 is followed by blocking AIM2 expression.Conclusion: (1) Ribosomal DNA accumulates in cfDNA of breast cancer patients; (2) Cell free rDNA induce DNA damage response and stimulates cells survival, including cells with an instable genome; (3) Cell free rDNA triggers TLR9- MyD88- NF-kB- signaling, with significantly repressing the expression of AIM2.

Estrogen receptor beta enhances chemotherapy response of GBM cells by down regulating DNA damage response pathways

Glioblastoma (GBM) is the most commonly diagnosed brain tumor that exhibit high mortality rate and chemotherapy resistance is a major clinical problem. Recent studies suggest that estrogen receptor beta (ERβ), may function as a tumor suppressor in GBM. However, the mechanism(s) by which ERβ contributes to GBM suppression and chemotherapy response remains unknown. We examined the role of ERβ in the DNA damage response of GBM cells, and tested whether ERβ sensitizes GBM cells to chemotherapy. Cell viability and survival assays using multiple epitope tagged ERβ expressing established and primary GBM cells demonstrated that ERβ sensitizes GBM cells to DNA damaging agents including temozolomide (TMZ). RNA-seq studies using ERβ overexpression models revealed downregulation of number of genes involved in DNA recombination and repair, ATM signaling and cell cycle check point control. Gene set enrichment analysis (GSEA) suggested that ERβ–modulated genes were correlated negatively with homologous recombination, mismatch repair and G2M checkpoint genes. Further, RT-qPCR analysis revealed that chemotherapy induced activation of cell cycle arrest and apoptosis genes were attenuated in ERβKO cells. Additionally, ERβ overexpressing cells had a higher number of γH2AX foci following TMZ treatment. Mechanistic studies showed that ERβ plays an important role in homologous recombination (HR) mediated repair and ERβ reduced expression and activation of ATM upon DNA damage. More importantly, GBM cells expressing ERβ had increased survival when compared to control GBM cells in orthotopic GBM models. ERβ overexpression further enhanced the survival of mice to TMZ therapy in both TMZ sensitive and TMZ resistant GBM models. Additionally, IHC analysis revealed that ERβ tumors had increased expression of γH2AX and cleaved caspase-3. Using ERβ-overexpression and ERβ-KO GBM model cells, we have provided the evidence that ERβ is required for optimal chemotherapy induced DNA damage response and apoptosis in GBM cells.

Heterogeneous Nuclear Ribonucleoproteins A1 and A2 Function in Telomerase-Dependent Maintenance of Telomeres.

The A/B subfamily of heterogeneous nuclear ribonucleoproteins (hnRNPs A/B), which includes hnRNP A1, A2/B1, and A3, plays an important role in cell proliferation. The simultaneous suppression of hnRNP A1/A2, but not the suppression of hnRNP A1 or A2 alone, has been shown to inhibit cell proliferation and induce apoptosis in cancer cells, but not in mortal normal cells. However, the molecular basis for such a differential inhibition of cell proliferation remains unknown. Here, we show that the simultaneous suppression of hnRNP A1 and hnRNP A2 resulted in dysfunctional telomeres and induced DNA damage responses in cancer cells. The inhibition of apoptosis did not alleviate the inhibition of cell proliferation nor the formation of dysfunctional telomeres in cancer cells depleted of hnRNP A1/A2. Moreover, while proliferation of mortal normal fibroblasts was not sensitive to the depletion of hnRNP A1/A2, the ectopic expression of hTERT in normal fibroblasts rendered these cells sensitive to proliferation inhibition, which was associated with the production of dysfunctional telomeres. Our study demonstrates that hnRNP A1 and A2 function to maintain telomeres in telomerase-expressing cells only, suggesting that the maintenance of functional telomeres in telomerase-expressing cancer cells employs factors that differ from those used in the telomerase-negative normal cells.

Nucleolar protein nucleolin functions in replication stress-induced DNA damage responses.

The nucleolus contains multiple copies of ribosomal (r)DNA, which indicate sites of frequent replication stress and suggest the existence of a mechanism to prevent replication stress–related rDNA instability and the possibility that such a mechanism contributes to the whole genomic stability against replication stress. We have previously reported that nucleolin, a major nucleolar protein, is involved in ionizing radiation–induced DNA damage responses (DDRs) such as ataxia telangiectasia mutated (ATM)-dependent cell cycle checkpoints and homologous recombination (HR) repair. Here, we investigated the role of nucleolin in DDR due to replication stress. The results indicate that following replication stress, nucleolin interacted with the histone γH2AX, proliferating cell nuclear antigen (PCNA), and replication protein A (RPA)32, suggesting that it may be recruited to DNA damage sites on the replication fork. Furthermore, the knockdown of nucleolin by siRNA reduced the activation of ATM and RAD3-related (ATR) kinase and the formation of RAD51 and RPA32 foci after replication stress due to UV or camptothecin exposure, whereas nucleolin overexpression augmented ATR-dependent phosphorylation and RAD51 and RPA accumulation on chromatin. Moreover, these overexpressing cells seemed to increase repair activity and resistance to replication stress. Our results indicate that nucleolin plays an important role in replication stress–induced DDRs such as ATR activation and HR repair. Given that nucleolin overexpression is often observed in many types of cancer cells, our findings suggest that nucleolin is involved in the regulation of resistance to replication stress that may otherwise lead to tumorigenesis and it could be a possible target for chemotherapy and radiotherapy.

Discovery of Isaindigotone Derivatives as Novel Bloom's Syndrome Protein (BLM) Helicase Inhibitors That Disrupt the BLM/DNA Interactions and Regulate the Homologous Recombination Repair.

Homologous recombination repair (HRR), a crucial approach in DNA damage repair, is an attractive target in cancer therapy and drug design. The Bloom syndrome protein (BLM) is a 3'-5' DNA helicase that performs an important role in HRR regulation. However, limited studies about BLM inhibitors and their biological effects have been reported. Here, we identified a class of isaindigotone derivatives as novel BLM inhibitors by synthesis, screening, and evaluating. Among them, compound 29 was found as an effective BLM inhibitor with a high binding affinity and good inhibitory effect on BLM. Cellular evaluation indicated that 29 effectively disrupted the recruitment of BLM at DNA double-strand break sites, promoted an accumulation of RAD51, and regulated the HRR process. Meanwhile, 29 significantly induced DNA damage responses, as well as apoptosis and proliferation arrest in cancer cells. Our finding provides a potential anticancer strategy based on interfering with BLM via small molecules.

In vitro genotoxicity analyses of colibactin-producing E. coli isolated from a Japanese colorectal cancer patient.

Colibactin is a polyketide-peptide genotoxin produced by enteric bacteria such as E. coli, and is considered to contribute to the development of colorectal cancer. We previously isolated E. coli strains from Japanese colorectal cancer patients, and in the present study we investigated the genotoxic potency of the colibactin-producing (clb+) E. coli strains that carry the polyketide synthases "pks" gene cluster (pks+) and an isogenic clb- mutant in which the colibactin-producing ability is impaired. Measurement of phosphorylated histone H2AX indicated that DNA double strand breaks were induced in mammalian CHO AA8 cells infected with the clb+ E. coli strains. Induction of DNA damage response (SOS response) by crude extract of the clb+ strains was 1.7 times higher than that of the clb- E. coli in an umu assay with a Salmonella typhimurium TA1535/pSK1002 tester strain. Micronucleus test with CHO AA8 cells revealed that infection with the clb+ strains induced genotoxicity, i.e., the frequencies of micronucleated cells infected with clb+ strain were 4-6 times higher than with the clb- strain. Since the intestinal flora are affected by dietary habits that are strongly associated with ethnicity, these data may contribute to both risk evaluation and prevention of colorectal cancer in the Japanese population.

The anticancer effects of MPT0G211, a novel HDAC6 inhibitor, combined with chemotherapeutic agents in human acute leukemia cells

BackgroundThere are some limitations of standard chemotherapy for acute leukemia. Vincristine and doxorubicin are commonly used for acute leukemia, but they may induce serious side effects such as cardiomyopathy and neurotoxicity. Furthermore, chemotherapy resistance occurs more and more frequently. Therefore, effective treatment strategies are needed. Histone deacetylase 6 inhibition is considered as a potential therapeutic strategy for acute leukemia, since it is observed that HDAC6 is overexpressed in acute leukemia and regulates tumor survival. Combination therapy for cancer is used to minimize adverse drug effects, reduce drug dosage, enhance efficacy, and prevent drug resistance. In order to improve efficacy of chemotherapy agents of acute leukemia, this study will investigate the effects of combination MPT0G211, a novel histone deacetylase 6 inhibitor, with doxorubicin or vincristine on human acute leukemia cells.ResultsMPT0G211 combined with doxorubicin induces DNA damage response on human acute myeloid leukemia cells. MPT0G211 can additionally increase Ku70 acetylation and release BAX to mitochondria. Ectopic expression of HDAC6 successively reversed the apoptosis triggered by the combined treatment. Moreover, co-treatment of MPT0G211 and vincristine may alter microtubule dynamics, triggering acute lymphoblastic leukemia cells arrest in mitotic phase followed by induction of the apoptotic pathway. Finally, MPT0G211 plus doxorubicin or vincristine can significantly improve the tumor growth delay in a tumor xenograft model.ConclusionsCollectively, our data highlighted that MPT0G211 in combination with chemotherapy drugs has significant anticancer activity, suggesting a novel strategy for the treatment of acute leukemia.

Free radical stress induces DNA damage response in RAW264.7 macrophages during Mycobacterium smegmatis infection

Genomic instability resulting from oxidative stress responses may be traced to chromosomal aberration. Oxidative stress suggests an imbalance between the systemic manifestation of reactive free radicals and biological system's ability to repair resulting DNA damage and chromosomal aberration. Bacterial infection associated insult is considered as one of the major factors leading to such stress conditions. To study free radical responses by host cells, RAW 264.7 macrophages were infected with non-pathogenic M. smegmatis mc2155 at different time points. The infection process was followed up with an assessment of free radical stress, cytokine, toll-like receptors (TLRs) and the resulting DNA damage profiles. Results of CFU count showed that maximum infection in macrophages was achieved after 9h of infection. Host responses to the infection across different time periods were validated from nitric oxide quantification and expression of iNOS and were plotted at regular intervals. IL-10 and TNF-α expression profile at protein and mRNA level showed a heightened pro-inflammatory response by host macrophages to combat M. smegmatis infection. The expression of TLR4, a receptor for recognition of mycobacteria, in infected macrophages reached the highest level at 9h of infection. Furthermore, comet tail length, micronuclei and γ-H2AX foci recorded the highest level at 9h of infection, pointing to the fact that breakage in DNA double strands in macrophage reaches its peak at 9h of infection. In contrast, treatment with ROS inhibitor N-acetyl-L-cysteine (NAC) prevented host cell death through reduction in oxidative stress and DNA damage response during M. smegmatis infection. Therefore, it can be concluded that enhanced oxidative stress response in M. smegmatis infected macrophages might be correlated with DNA damage response.

Co-Localization Analysis Method for High Content Screening (HCS) Measurement of Radiation Induced DNA Damage Response

Co-Localization Analysis Method for High Content Screening (HCS) Measurement of Radiation Induced DNA Damage Response

1901P - ATM as a DNA damage response protein in uveal melanoma: Association with clinicopathological factors and prognostic outcome

Background: Uveal melanoma (UM) is an intraocular malignancy commonly arising from choroid which can cause visual loss or metastasis. Till date, there is no current study available on UM with respect to ATM (Ataxia Telangiectasia Mutated) protein that induces DNA damage response. Several studies revealed that loss of nuclear ATM (nATM) in various cancers like pancreatic, colorectal, gastric cancer leads to poor prognosis. This signifies ATM protein as a prognostic biomarker for cancer progression. Therefore, the aim of the study is to detect the expression/localization of ATM protein in uveal melanoma patients. Methods: Expression of nATM was investigated on 69 formalin fixed paraffin embedded choroidal melanoma samples by immunohistochemistry and validated by western blotting. Results were then correlated with clinical and histopathological parameters. To determine the prognostic significance, Kaplan–Meier analysis and multivariate analysis by Cox’s Proportional Hazards Model was performed. Results: There was a male preponderance in our study. Histopathological high-risk factors were identified in 30/69 (43.5%) cases. Loss of nATM was found in 65.2% of the cases. Loss of nATM was statistically significant with epithelioid cell type, high pigmentation, LTD >10mm, HRFs>1, tumour height and advanced tumour staging (p < 0.05). On multivariate analysis, advanced tumour staging found out to be an independent prognostic factor. Conclusions: Our data suggest that loss of nATM protein might serve as a potential prognostic marker in the pathogenesis of uveal melanoma and leads to increased risk of metastasis. These findings demonstrate an important role of ATM protein and may have a therapeutic potential in uveal melanoma. However, further studies are required in a larger cohort of patients with longer follow up and translational validation needs to be performed. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Abstract 4839: Targeting glioma stem cells by pharmacologic stabilization of G-quadruplexes

Abstract Among heterogeneous cell population in a tumor, cancer stem cells (or tumor-propagating cells) are defined as a cell fraction that shows self-renewal, multipotency, high tumorigenicity, and resistance to chemo/radiotherapy. Because cancer stem cells are supposed to drive initiation, metastasis, and recurrence of the disease, identification of their therapeutic targets would have a profound implication for eradication of cancer. G-quadruplex (G4) is an atypical four-stranded nucleic acid structure that can be formed at guanine-rich sequences, such as the telomeric TTAGGG repeats. Accumulating evidence supports that G4s are widely distributed through the genome in living cells and affect various intracellular events, including DNA replication, gene expression and translation, whereas pharmacologic stabilization of G4s has been implicated for cancer intervention. However, precise mechanisms for the efficacy and target cancer types remain elusive. Here we demonstrate that G4 stabilization by chemical compounds, called G4 ligands, preferentially inhibits the growth of glioma stem cells in culture and in vivo. While the established glioma stem cells maintain stemness under the serum-free sphere culture conditions, serum stimulation induces their differentiation into non-stem glioma cells. We found that these glioma stem cells are highly sensitive to G4 ligands, such as a natural compound telomestatin and its newly synthesized derivative, and a bisquinolinium compound Phen-DC3, all of which can stabilize G4s. Upon the ligand treatment, glioma stem cells activated the replication stress pathway more potently than non-stem glioma cells, which could partly explain the selectivity of the deleterious effect. Consistently, these ligands induced DNA damage response in glioma stem cells but not in non-stem glioma cells. In glioma stem cells, about 20% to 30% of DNA damage foci were telomeric (i.e., telomere dysfunction-induced foci: TIFs) whereas the rest were non-telomeric, suggesting that the G4 ligands recognized both telomeric and non-telomeric G4s. Temozolomide, a clinical alkylating agent for brain tumors, induced DNA damage response but did not preferentially attack telomeres. As a potential pharmacodynamic biomarker, the immunofluorescence intensities of the nuclear G4 foci were enhanced by G4 ligands. Furthermore, these ligands directly inhibited in vitro transcription and translation of mRNAs that contained G4-forming sequences, suggesting additional mode of action. While G4 ligands inhibit telomerase activity, classical telomerase inhibitors without G4-stabilizing activity had no preferential impact on glioma stem cells. Together, these observations suggest that G4 is a promising target for pinpointing intractable glioma stem cells. Citation Format: Sachiko Okabe, Takahiro Nakamura, Daiki Hasegawa, Reina Kojima, Keiji Okamoto, Ichiro Nakano, Kazuo Shin-ya, Kazuo Nagasawa, Hiroyuki Seimiya. Targeting glioma stem cells by pharmacologic stabilization of G-quadruplexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4839.

The live cell DNA stain SiR-Hoechst induces DNA damage responses and impairs cell cycle progression

SiR-Hoechst (SiR-DNA) is a far-red fluorescent DNA probe being used widely for time-lapse imaging of living cells that is reported to be minimally toxic at concentrations as high as 10–25 µM. However, measuring nuclear import of Cyclin B1, inhibition of mitotic entry, and the induction of γH2AX foci in cultured human cells reveals that SiR-Hoechst induces DNA damage responses and G2 arrest at concentrations well below 1 µM. SiR-Hoechst is useful for live cell imaging, but it should be used with caution and at the lowest practicable concentration.

Anticancer activity of arborinine from Glycosmis parva leaf extract in human cervical cancer cells

Glycosmis parva is a small shrub found in Thailand. Ethyl acetate (EtOAc) extract from its leaves has been shown to exert anticancer effects in vitro; however, the compound responsible for this activity has not been isolated and characterized. In this study, we demonstrate that arborinine, a major acridone alkaloid in the EtOAc fraction, decreased proliferation and was strongly cytotoxic to HeLa cervical cancer cells without significantly affecting normal cells. The compound also inhibited tumor spheroid growth much more potently than chemotherapeutic drugs bleomycin, gemcitabine, and cisplatin. In addition, unlike cisplatin, arborinine activated caspase-dependent apoptosis without inducing DNA damage response. We further show that arborinine strongly suppressed cancer cell migration by downregulating expression of key regulators of epithelial-mesenchymal transition. Taken together, our data provide important insights into the molecular mechanism of arborinine’s anticancer activity, supporting its potential use for treating cervical cancer.

P-339 - Modulation of redox homeostasis in grade IV glioblastoma multiforme

A carbazole alkaloid, mahanine, isolated from an Indian Medicinal plant enhances intracellular reactive oxygen species (ROS) by the inhibition of complex-III of mitochondrial electron transport chain (ETC). Elevated ROS mediates apoptosis by damaging cellular homeostasis in several malignancies with various genetic mutations in multidimensional facets. We observed oxidative damage-induced dysfunction of oncogenic chaperone Hsp90 in pancreatic cancer, leukemia and activation of PTEN in colon cancer. This accumulated ROS induced DNA damage response, that mediated Chk1/Chk2 upregulation and activation which were essential factors for the G0/G1 arrest in glioblastoma multiforme (GBM) without effecting normal astrocytes. During G0/G1 arrest, cyclin D1/cyclin D3, CDK4/CDK6 and CDC25A were also downregulated. Mahanine-induced ROS in hypoxia responsible for higher G0/G1 arrest. Mahanine-treated G0/G1 arrested cells were less potent to form xenograft tumor. They exhibited reduced ability to migrate and form intracellular tubelike structures and became susceptible to differentiation. Astrocyte-like cells were generated from the epithelial lineage. Thus, we established that complex-III of ETC is one of the possible potential targets of this nontoxic chemotherapeutic molecule.

Cyclic Peptidic Mimetics of Apollo Peptides Targeting Telomeric Repeat Binding Factor 2 (TRF2) and Apollo Interaction.

Telomeric repeat binding factor 2 (TRF2) is a telomere-associated protein that plays an important role in the formation of the 3' single strand DNA overhang and the "T loop", two structures critical for the stability of the telomeres. Apollo is a 5'-exonuclease recruited by TRF2 to the telomere and contributes to the formation of the 3' single strand DNA overhang. Knocking down of Apollo can induce DNA damage response similar to that caused by the knocking down of TRF2. In this Letter, we report the design and synthesis of a class of cyclic peptidic mimetics of the TRFH binding motif of Apollo (ApolloTBM). We found conformational control of the C terminal residues of ApolloTBM can effectively improve the binding affinity. We have obtained a crystal structure of a cyclic peptidic Apollo peptide mimetic (34) complexed with TRF2, which provides valuable guidance to the future design of TRF2 inhibitors.

TRIM8 regulated autophagy modulates the level of cleaved Caspase-3 subunit to inhibit genotoxic stress induced cell death

In cancer patients, treatment modalities like chemotherapy and radiation exert their anticancer effects by inducing DNA damage. The cancer cells can survive under genotoxic stress by inducing DNA damage response (DDR) or can undergo cell death. The process of autophagy is emerging as crucial regulator of cell survival during different stress conditions. Post translational modification through ubiquitin plays an essential role in DDR during genotoxic stress conditions. Ubiquitin ligases regulate autophagy and cell death pathways however their role during genotoxic stress conditions is not understood. In the current study we identified TRIM8, RING E3 Ligase, as a novel regulator of autophagy during DDR. TRIM8 regulates lysosomal biogenesis and autophagy flux. The turnover of TRIM8 is high and is stabilized during genotoxic stress conditions. TRIM8 regulated autophagy is essential for its cytoprotective role during genotoxic stress induced cell death. TRIM8 stabilizes the turnover of XIAP during genotoxic stress and forms complex with XIAP and caspase-3 to inhibit its activation in presence of etoposide. TRIM8 mediated autophagy promotes degradation of cleaved caspase-3 subunits. This study described TRIM8, as a novel regulator of DDR-autophagy crosstalk, which may play role in survival of cancer cells in presence of genotoxic agents.

The Novel Small Molecule STK899704 Promotes Senescence of the Human A549 NSCLC Cells by Inducing DNA Damage Responses and Cell Cycle Arrest.

The novel synthetic compound designated STK899704 (PubChem CID: 5455708) suppresses the proliferation of a broad range of cancer cell types. However, the details of its effect on lung cancer cells are unclear. We investigated the precise anticancer effect of STK899704 on senescence and growth arrest of A549 human non-small cell lung cancer (NSCLC) cells. STK899704 affected NSCLC cell cycle progression and decreased cell viability in a dose-dependent manner. Immunofluorescence staining revealed that STK899704 destabilized microtubules. Cell cycle analysis showed an increase in the population of NSCLC cells in the sub-G1 and G2/M phases, indicating that STK899704 might cause DNA damage via tubulin aggregation. Furthermore, we observed increased mitotic catastrophe in STK899704-treated cells. As STK899704 led to elevated levels of the p53 pathway-associated proteins, it would likely affect the core DNA damage response pathway. Moreover, STK899704 promoted senescence of NSCLC cells by inducing the p53-associated DNA damage response pathways. These findings suggest that the novel anti-proliferative small molecule STK899704 promotes cell death by inducing DNA damage response pathways and senescence after cell cycle arrest, being a potential drug for treating human lung cancers.