bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3006: Hyperoxia may be a treatment option for NSCLC
Authors: Hamamoto Junko , Yasuda Hiroyuki , Nakachi Ichiro , Edwards Michael G. , Miyawaki Masayoshi , Nishino Makoto , Kuroda Aoi , Tani Tetsuo , Arai Daisuke , Ishioka Kota , Kawada Ichiro , Naoki Katsuhiko , Betsuyaku Tomoko , Soejima Kenzo
Published: August 2015

Introduction: Many anti-tumor drugs have been developed, however, prognosis of NSCLC remains poor and new approaches for NSCLC treatment are expected. Lung is a unique organ whose epithelial cells are directly exposed to oxygen. Hyperoxia produces reactive oxygen species (ROS) and induces cell damage. Therefore we attempted to investigate the potential of hyperoxia as an anti-tumor treatment in NSCLC cells in this study.

Methods: We used oxygen-concentration-adjustable incubators and validated effect of hyperoxia on various lung cancer cell lines in vitro. The effect of hyperoxia on cell proliferation, apoptosis and gene expression was evaluated using MTS assay, flowcytometry and cDNA microarray, respectively.

Results: We found that the growth of lung cancer cell lines (A549, NCI-H1975) was inhibited by 50% of hyperoxia in a dose-dependent manner, while that of normal airway epithelial cells (NHBE, BEAS-2B) was not. Two independent pathway analysis using cDNA microarray revealed the activation of NF-kB and AMPK pathways, and the production of nitric oxide and ROS in hyperoxia (50%) treated lung cancer cell lines compared to untreated ones. The combined treatment with ABT-263, Bcl-2 inhibitor, and hyperoxia (50%) induced synergistic growth inhibition and apoptosis in NCI-H1975 and SK-MES-1 cells. Moreover, the combination of ABT-263 and activator of either ROS, NF-kB or AMPK also showed synergistic growth inhibition in those cells.

Conclusions: Hyperoxia (50%) showed anti-tumor effect in NSCLC cell lines through the activation of NF-kB and AMPK pathways, and the production of ROS. The effect was augumented in combination with Bcl-2 inhibitor. Hyperoxia may be a treatment option for NSCLC patients.

Citation Format: Junko Hamamoto, Hiroyuki Yasuda, Ichiro Nakachi, Michael G. Edwards, Masayoshi Miyawaki, Makoto Nishino, Aoi Kuroda, Tetsuo Tani, Daisuke Arai, Kota Ishioka, Ichiro Kawada, Katsuhiko Naoki, Tomoko Betsuyaku, Kenzo Soejima. Hyperoxia may be a treatment option for NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3006. doi:10.1158/1538-7445.AM2015-3006
Abstract 3014: Identification of the NDP kinase critical for DNA repair
Authors: Tsao Ning , Deng Yu-Jyun , Chang Zee-Fen , Zhang Wei
Published: August 2015

Introduction and Purpose:

Cellular supply of deoxynucleoside triphosphates (dNTPs) is crucial for DNA replication and repair. It is known that ribonucleotide reductase (RNR), a rate-limiting enzyme for dNTP synthesis, is required for DNA repair. The catalytic function of RNR produces dNDPs. There are eight isoforms of NDP kinase that catalyzes the subsequent step for dNTP formation in human cells. In this study, we aim to identify the isoform of NDP kinase required for DNA repair.

Preliminary results:

By RNA interference experiments, we identified NME3, one isoform of the nucleoside diphosphate kinases, critical for DNA repair. It has been shown that RNR forms a complex with Tip60, a histone acetyltransferase, by which RNR is recruited to DNA damage sites for repair. In this study, we found that NME3 directly interacts with Tip60. The effect of knockdown of NME3 on DNA repair was rescued by re-expression of wild-type but not the mutant type defective in Tip60 interaction. Overexpression of NME3 was able to facilitate DNA repair, which was intervened by disrupting the interaction of NME3 with Tip60. Moreover, exogenous addition of deoxynucleosides facilitates NME3-mediated DNA repair.

Conclusion:

Among 8 isoforms of NDP kinase, NME3 is identified to be critical for DNA repair, indicating its potential as a target for developing chemosensitization agent.

Citation Format: Ning Tsao, Yu-Jyun Deng, Zee-Fen Chang, Wei Zhang. Identification of the NDP kinase critical for DNA repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3014. doi:10.1158/1538-7445.AM2015-3014
Abstract 3015: BCL10 is a novel DNA damage response factor whose expression inversely correlates with the clinical outcome of breast invasive ductal carcinoma
Authors: Zhu Min , Zhao Hongchang , Xu Xingzhi
Published: August 2015

Timely and properly cellular response to DNA damage is essential for maintenance of genome stability and integrity. B-cell lymphoma/leukemia 10 (BCL10) facilitates ubiquitination of NEMO in the cytosol, activating NFκB signaling. Translocation and/or point mutations of BCL10 associate with mucosa-associated lymphoid tissue lymphomas and other malignancies. We recently found that BCL10 in nucleus is enriched onto the DNA damage sites in an ATM- and RNF8-dependent manner, and ATM-dependent phosphorylation and RNF8-dependent ubiquitination of BCL10 confers UBC13 to RNF8/RNF168 to regulate ubiquitination-mediated DSB signaling and repair. In this report, we further found that inhibition of BCL10 expression compromised RNF168 dimerization and RNF168-dependent histone H2A ubiquitination. Immunohistochemistry staining analysis revealed that BCL10 expression status inversely correlates with prognosis in 339 cases of breast invasive ductal carcinoma with a median follow up of 60 months (range, 3-127 months). Taken together, our results demonstrated that BCL10 is a novel DDR factor with a potential role as a biomarker in breast cancer therapy.

Note: This abstract was not presented at the meeting.

Citation Format: Min Zhu, Hongchang Zhao, Xingzhi Xu. BCL10 is a novel DNA damage response factor whose expression inversely correlates with the clinical outcome of breast invasive ductal carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3015. doi:10.1158/1538-7445.AM2015-3015
Abstract 3017: Ribonucleotide reductase promotes uracil-mediated genome instability in tumor progression
Authors: Chen Chih-Wei , Tsao Ning , Yen Yun , Lehman Christine E. , Wang Yuh-Hwa , Chen Chian-Feng , Wu Tse-Hsiang , Tien Sui-Chih , Lee Ming-Hsiang , Chang Zee-Fen
Published: August 2015

The elevation of RRM2A subunit of ribonucleotide reductase (RNR) has been shown oncogenic and associated with human cancers. However, the molecular mechanism of RRM2A in cancer promotion is unclear. In this study, we investigated whether RNR-mediated dUTP formation is involved in promoting genome instability. Since dUTP is degraded by dUTPase, we analyzed the expression of RRM2A and dUTPase in clinical samples from colorectal and breast cancer patients. The results revealed that high RRM2A and low dUTPase in tumors are correlated with poorer survival. In cancer cell lines that express high level of RRM2A and dUTPase, knockdown of dUTPase increases genome instability. Conversely, overexpression of dUTPase reduces genome stress in cancer cells expressing high RRM2A and low dUTPase. In non-tumorigenic cells, overexpression of RRM2A increases replication stress and genome instability accompanied by higher levels of 53BP1 foci and increased breaks at AT-rich fragile sites bound by 53BP1. Thymidine supplement in growth medium or co-expression of dUTPase was able to reduce RRM2A-mediated replication stress and 53BP1 foci. We further provide evidence that RRM2A promotes replication stress and error prone-translesion synthesis dependent on uracil DNA glycosylase. These results suggest that up-regulation of RRM2A in tumor cells increases uracil-mediated replication stress to promote genome instability. Therefore, high RRM2A/low dUTPase context confers tumor genome evolution advantage, correlating with poorer prognosis.

Citation Format: Chih-Wei Chen, Ning Tsao, Yun Yen, Christine E. Lehman, Yuh-Hwa Wang, Chian-Feng Chen, Tse-Hsiang Wu, Sui-Chih Tien, Ming-Hsiang Lee, Zee-Fen Chang. Ribonucleotide reductase promotes uracil-mediated genome instability in tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3017. doi:10.1158/1538-7445.AM2015-3017
Abstract 3024: Lack of CK1δ increases DNA damage and genomic instability due to defects in DNA repair and mitotic checkpoints
Authors: Greer Yoshimi E. , Gao Bo , Yang Yingzi , Lipkowitz Stanley , Rubin Jeff S.
Published: August 2015

Casein kinase 1 delta (CK1δ) is a conserved serine/threonine protein kinase that regulates diverse cellular processes including vesicle trafficking and circadian rhythm. We previously reported that CK1δ is a mediator of Wnt-3a-dependent neurite outgrowth (Greer and Rubin, JCB, 2011) and ciliogenesis (Greer et al. MBoC, 2014). Mice that lack Csnk1d exhibit a perinatal lethal phenotype and typically weigh 30-50% less than their wild type littermates, however, the exact causes of death and small size are unknown. We hypothesized that the absence of CK1δ contributes to cellular stresses that adversely affect cell survival. Mouse embryonic fibroblasts (MEFs) were collected from mice homozygous for a Csnk1d floxed allele, and MEFCsnk1d null cells were generated by infection with adenovirus (Ad) expressing Cre. As control, MEFCtl. cells were produced by infection with Ad-GFP. In addition, MEFCtl cells were treated with CK1δ siRNA reagents to transiently suppress endogenous expression. Cells were analyzed by confocal microscopy, western blotting and flow cytometry. At early passage (P2), many MEFCsnk1d null cells had detached from the culture dish, raising the possibility of cell death. Subsequent MTS assay confirmed a significantly lower cell viability of MEFCsnk1d null cells compared to MEFCtl cells. MEFCsnk1d null cells (P3) exhibited a larger sub-Go fraction (64%) than MEFCtl cells (34%) in flow cytometric analysis. γ-H2AX staining and comet assay confirmed significant DNA damage in MEFCsnk1d null cells. MEFCsnk1d null cells exhibited micronuclei, a characteristic of genomic instability. Similarly, abrogation of CK1δ expression in MEFCtl cells induced micronuclei formation in response to DNA damage triggered by doxorubicin, suggesting that the absence of CK1δ increases susceptibility to genotoxic exposure. Chk1, a kinase with a major role in DNA damage response, and its downstream substrate, Cdc2/CDK1 kinase, were inhibited in MEFCsnk1d null cells as well as in MEFCtl cells transfected with CK1δ siRNA, suggesting that CK1δ is required for the G2/M mitotic checkpoint. Hydroxyurea-induced Chk1 activation was also impaired in cells lacking CK1δ, and re-expression of CK1δ rescued the phenotype. Together, the data indicate that CK1δ is required for efficient repair of DNA damage and the proper functioning of mitotic checkpoints. The mechanism(s) responsible for these activities are currently under investigation.

Citation Format: Yoshimi E. Greer, Bo Gao, Yingzi Yang, Stanley Lipkowitz, Jeff S. Rubin. Lack of CK1δ increases DNA damage and genomic instability due to defects in DNA repair and mitotic checkpoints. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3024. doi:10.1158/1538-7445.AM2015-3024
Abstract 3032: Genomic deregulation and therapeutic role of the cell-cycle kinase TLK2 in more aggressive breast cancers
Authors: KIM Jin-Ah , Tan Ying , Wang Xian , Cao Xixi , Veeraraghavan Jamunarani , Liang Yulong , Edwards Dean P. , Pan Xuewen , Li Kaiyi , Schiff Rachel , Wang Xiaosong
Published: August 2015

More aggressive breast cancers often coincide with enhanced chromosomal instability (CIN), which poses a great challenge to clinical management. By integrative analysis of genomic data, we observed the amplification of tousled-like kinase 2 (TLK2), a cell cycle serine-threonine kinase, in approximately 9% of breast cancer, which is more frequent in the luminal B and late-stage breast tumors. The resulting TLK2 upregulation correlates with the level of CIN in breast tumors, and predicts worse outcome regardless of endocrine therapy. Further studies suggest that TLK2 overexpression leads to a G2/M checkpoint defect, delayed DNA repair, and enhanced invasiveness. TLK2 silencing selectively inhibits the growth of TLK2-high breast cancer cells, and induces apoptosis. This response is retained in the breast cancer cells with acquired endocrine resistance. TLK2 inhibition in a preclinical tumor model significantly improved progression-free survival. Together, targeting TLK2 presents an attractive therapeutic strategy on TLK2-amplified breast cancers.

Citation Format: Jin-Ah KIM, Ying Tan, Xian Wang, Xixi Cao, Jamunarani Veeraraghavan, Yulong Liang, Dean P. Edwards, Xuewen Pan, Kaiyi Li, Rachel Schiff, Xiaosong Wang. Genomic deregulation and therapeutic role of the cell-cycle kinase TLK2 in more aggressive breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3032. doi:10.1158/1538-7445.AM2015-3032
Abstract 3043: Discovery of a novel mitochondrial protein complex containing pro-apoptotic Noxa in leukemia
Authors: Gaynes Jeffrey S. , Hanse Eric A. , Kelekar Ameeta
Published: August 2015

Apoptosis evasion and altered nutrient metabolism are recognized as major contributors to cancer development. However, the degree to which apoptosis and metabolism are integrated in malignant cells remains poorly understood. Previously our group observed alterations in mitochondrial function mediated by expression of the pro-apoptotic member of the Bcl-2 family Noxa. Here, we report the presence of a large 650 kilodalton multi-protein complex specific to the mitochondria of Jurkat leukemia cells that contains Noxa. We also detect a similar mitochondrial protein complex in cells expressing Noxa with a mutated BH3 domain, suggestive of an apoptosis-independent metabolic function for this Bcl-2 family member. Lastly, we present here the successful isolation of this complex aimed at identifying other complex components via mass spectrometry. This unique strategy included purifying the complex by gel-filtration chromatography, and then resolving the complex into an acrylamide gel by either size or charge, followed by separating the individual complex components by size. Future studies are geared towards identifying the other components of the complex, looking for presence of this complex in other cancers, and understanding how this complex may function in maintaining mitochondrial homeostasis in cancer. In summary, the data presented here strongly suggests a non-canonical function for Noxa in regulating mitochondrial function independent of apoptosis in leukemia, and possibly other malignancies.

Note: This abstract was not presented at the meeting.

Citation Format: Jeffrey S. Gaynes, Eric A. Hanse, Ameeta Kelekar. Discovery of a novel mitochondrial protein complex containing pro-apoptotic Noxa in leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3043. doi:10.1158/1538-7445.AM2015-3043
Abstract 3058: Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents
Authors: Kumar Sandeep , Yadav Neelu , Marlowe Tim , Chaudhary Ajay , Wang Jianmin , O'Malley Jordan , Boland Patrick , Jayanthi Srinivas , Kumar Thallapuranam Krishnaswamy Suresh , Yadava Nagendra , Chandra Dhyan
Published: August 2015

Although cancer cells develop resistance to multiple types of anticancer agents, whether they adopt similar or differential mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. We show differential sensitivity, caspase activation, and cytokines/chemokines release in response to multiple anticancer agents. We demonstrated that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induced apoptosis in OXPHOS-independent manner. DNA-damaging agents induced mitochondrial biogenesis accompanied by increased production of cellular and mitochondrial ROS. Mitochondrial biogenesis upregulated both mitochondrial protein-folding machinery and unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA damage-induced caspase activation, whereas inhibition of complex II or gross complexes did not modulate the caspase activity. Interestingly, DNA damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted upon OXPHOS complex I-deficiency but not by OXPHOS complex II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross-complexes whereas a reverse trend was observed with apicidin. Together, our findings define the specific targets of apoptosis induction in response to a broad range of anticancer therapeutics, which provide a new strategy for differential mitochondrial targeting for cancer therapy.

Citation Format: Sandeep Kumar, Neelu Yadav, Tim Marlowe, Ajay Chaudhary, Jianmin Wang, Jordan O'Malley, Patrick Boland, Srinivas Jayanthi, Thallapuranam Krishnaswamy Suresh Kumar, Nagendra Yadava, Dhyan Chandra. Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3058. doi:10.1158/1538-7445.AM2015-3058
Abstract 3068: Integrative tcga analyses identify key microRNAs involved in chemoresistance
Authors: Shen Fangrong , Vasudha Sehgal , Wu Sherry , Ehsan A Ehsanipour , Robert C Olson , Cristina Ivan , Keith A Baggerly , Prahlad Ram , Anil K Sood
Published: August 2015

Objective. High-grade serous ovarian cancer (HGSOC) is the most common and lethal type of ovarian carcinoma. Our study aims to identify key microRNAs (miRs) that are responsible for promoting chemoresistance in HGSOC. Methods. We developed fast and robust computational algorithm to identify miRs that are significantly associated with improved or worsened survival in HGSOC patients. We subsequently used this algorithm to mine The Cancer Genome Atlas (TCGA) ovarian cancer database to identify miRs that are up-regulated in tumors from patients whose cancer had progressed within 6 months of diagnosis (chemoresistant group) compared to tumors from patients who had no disease progression for more than 2 years (chemosensitive group). We also constructed miRNA-mRNA functional networks for the key miRs identified. The biological roles of selected miRs were subsequently examined in multiple chemoresistant (SKOV3-TR, HeyA8-MDR, and A2780-CP20) ovarian cancer cell lines. Results. From our integrative analyses, we identified several miRs being highly up-regulated or down-regulated in chemoresistant HGSOC (p<0.05, top 10%). To examine the functional roles of these miRs, we transfected miR mimics and anti-miR constructs into taxane or platinum resistant cell lines. We showed that anti-miR-1271 significantly enhanced the sensitivity of taxane-resistant cancer cells (SKOV3-TR and HeyA8-MDR) to paclitaxel and docetaxel treatments. In contrast, transfecting anti-miR-1271 did not alter the sensitivity of A2780-CP20 cells to cisplatin treatment. Conclusion. These data provide the first integrated and functional approach for overcoming chemoresistance using miRs.

Citation Format: Fangrong Shen, Sehgal Vasudha, Sherry Wu, Ehsanipour, Ehsan A, Olson Robert C, Ivan, Cristina, Baggerly, Keith A, Ram, Prahlad, Sood, Anil K. Integrative tcga analyses identify key microRNAs involved in chemoresistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3068. doi:10.1158/1538-7445.AM2015-3068
Abstract 3069: The role of miR-30c-2* in clinical outcome and drug resistance in HPV-infected non-small cell lung cancer
Authors: Cheng Ya-Wen , Lin Hsiao-Ching
Published: August 2015

Lung cancer is leading cause of cancer death in Taiwanese women who are mostly to be life-time never smokers. Majority of drugs and combinations are used to treat with smoking lung cancer patients, not for nonsmokers. However, the 5-year survival rate in lung cancer patients remains ∼15% during the past three decades. Therefore, dissolving tumor recurrence and drug resistance is urgent needed for improving outcome in lung cancer, especially in nonsmokers. Mir-30c-2* has been considered to be tumor suppressor gene in various cancers. MiR-30c-2* levels were associated with in gemcitabine sensitivity of lung cancer cells. Down-regulation of miR-30c promotes tumor invasion via an increased in MTA1 expression. Our previous reports have indicated that HPV16/18 infection may be involved in Taiwanese lung tumorigenesis. Preliminary data showed that miR-30C-2* levels were elevated 45-fold in E6-knockdown TL-1 cells as compared with parental cells with non-specific RNAi tranfection. More interestingly, MTA-1 expression was negatively correlated with miR-30C-2* in lung tumors from lung cancer patients. Expression levels of MTA-1 were positive correlated with tumor stage and nodal metastasis in tumor tissues of lung cancer patients. Our cell model studies also found that miR-30C-2* suppressed by E6 could contribute to tumor metastasis and drug resistance via an increased in MTA-1 expression. These results were showed that miR-30C-2* levels in patients’ tumor tissues could be useful to predict outcome and therapeutic response and to select useful therapy drugs for lung cancer patients, especially in patients with HPV-infection.

Citation Format: Ya-Wen Cheng, Hsiao-Ching Lin. The role of miR-30c-2* in clinical outcome and drug resistance in HPV-infected non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3069. doi:10.1158/1538-7445.AM2015-3069
Abstract 3078: Epigenetic regulation of miR-138 confers cancer stem cell characteristics of renal cell carcinoma
Authors: Yun Eun-Jin , Zhou Jiancheng , Lin Chun-Jung , Hernandez Elizabeth , Santoyo John , Hsieh Jer-Tsong
Published: August 2015

Chemotherapy is the standard care for metastatic disease, in renal cell carcinoma (RCC), the outcome of chemotherapy only prolongs short survival of patients. The reason for the resistance of RCC to chemotherapy is not fully understood, although it has been speculated that overabundance of ATP-binding cassette (ABC)/p-glycoprotein transporter system in RCC might be the reason to repel various chemotherapeutic agents away from the cancer cell. Meanwhile, cancer stem cells (CSC) theory has also focused a significant attention in recent years as a potential explanation for drug resistance and metastasis of RCC. In this study, we demonstrate that drug resistant RCC cells exhibit CSC phenotypes and miR-138 that is significantly downregulated in RCC acted critical role in suppressing CSC phenotype by targeting ABC transporter ABCA13 and oncogenic histone methyltransferase EZH2. Importantly, miR-138 levels were significantly enhanced by treatment of DNA hypomethylating agent (i.e., 5-aza) or histone deactylases inhibitor (i.e., TSA). Epigenetic silencing of tumor-suppressive micro-RNAs has been shown a key oncogenic mechanism for the activation of oncogenes in tumors. Indeed, depletion of miR-138 promoted CSC phenotype followed by enhanced expressions of ABCA13 and EZH2. Furthermore, a novel tumor suppressor DAB2IP can inhibit DNA methyltransferase 1 (DNMT1) activity, which results in miR-138 expression. Taken together, this study identifies the new role of miR-138 in RCC-CSC and unveils a unique regulation of tumor suppressor protein in DNMT1. Overall study provides not only a new biomarker of RCC-CSC and but also potential therapeutic strategy of targeting drug resistant RCC.

Citation Format: Eun-Jin Yun, Jiancheng Zhou, Chun-Jung Lin, Elizabeth Hernandez, John Santoyo, Jer-Tsong Hsieh. Epigenetic regulation of miR-138 confers cancer stem cell characteristics of renal cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3078. doi:10.1158/1538-7445.AM2015-3078
Abstract 3080: Definition of the Ewing sarcoma specific oncomir-1 targetome
Authors: Schwentner Raphaela , Herrero-Martin David , Kauer Maximilian , Kovar Heinrich
Published: August 2015

MicroRNAs (miRNA) serve to fine-tune gene expression and thus play an important regulatory role in tissue specific gene networks. The identification and validation of miRNA target genes in a given tissue still poses a significant problem, since the presence of a seed sequence in the 3´UTR of an mRNA and its expression modulation upon forced ectopic expression of the miRNA do not reliably predict regulation under physiological conditions. Here, we used a combination of AGO2 pull-down experiments by PAR-CLIP - to enrich and sequence RISC-associated RNAs - and of RNAseq upon miRNA depletion by ectopic sponge expression, to identify the targetome of miR17-92 in Ewing sarcoma (ES) cells. The chimeric oncoprotein EWS-FLI1 is the driving pathogenic force in ES. As an aberrant ETS transcription factor it directly deregulates a multitude of genes related to proliferation control and differentiation. Recently, we reported on the EWS-FLI1 microRNA (miRNA) signature based on knockdown experiments in ES cell lines and on differential expression in primary tumors versus mesenchymal stem cells. Comparison of both datasets revealed miR20a-3p to be the top EWS-FLI1 activated miRNA. MiR-20a belongs to the miR17-92 cluster, also known as oncomir-1, which is one of the most potent oncogenic miRNAs. The whole cluster is among the small number of miRNAs down-regulated upon knockdown of EWS-FLI1 in multiple ES cell lines. Since feedback loops exists between the E2F transcription factor family and miR-17-92 and between EWS-FLI1 and E2F3, we sought to investigate the role of this miRNA cluster in ES.

We found a significant enrichment of PAR-CLIP hits for members of the miR-17-92 cluster, in the 3´UTRs of genes up-regulated in response to mir-17-92 specific sponge expression. Among them we consistently identified known (i.e. CTGF, MXD1, CYLD1) and a multitude of so far unknown targets of the oncomir-1 cluster in ES in three PAR-CLIP experiments. Preliminary experiments using 3′UTRs of select novel candidate genes in gene reporter assays showed up-regulation of luciferase activity upon transfection of a sponge specific to miR-17-92 and 20a, but not to unrelated miR-9 and 10b. Site directed mutagenesis of seed sequences in the 3′UTR of these candidate genes will be used for further validation.

This study provides an example for the comprehensive definition of the targetome for a defined group of miRNAs under physiological conditions.

This study was supported by the 7th framework program of the European Commission (FP7-259348, “ASSET”) and the Austrian Science Fund FWF (24708-B21)

Citation Format: Raphaela Schwentner, David Herrero-Martin, Maximilian Kauer, Heinrich Kovar. Definition of the Ewing sarcoma specific oncomir-1 targetome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3080. doi:10.1158/1538-7445.AM2015-3080
Abstract 3096: CDK4/6 as a therapeutic target in malignant melanoma
Authors: Teh Jessica L. , Aplin Andrew
Published: August 2015

Melanoma is the most deadly form of skin cancer and FDA-approved therapies such as vemurafenib and ipilimumab are associated with short-term responses and/or severe toxicities for patients. Recent favorable clinical data with cyclin-dependent kinase (CDK) 4/6 inhibitor, palbociclib (PD’991) in ER+ breast cancer has rekindled an interest in targeting cell cycle progression to stop aberrant cell growth. Thus, we set out to analyze the potential use of CDK4/6 inhibitors in melanoma. Due to the inability of CDK4/6 inhibitors to induce apoptosis as a single agent, we explored viable combinatorial strategies that will trigger both pro-senescent and pro-apoptotic pathways that may lead to an effective treatment modality for melanoma patients. Here, we demonstrate the benefit of combining a MEK inhibitor, trametinib (GSK’212) with PD’991 both in vitro and in vivo. We did not observe any enhanced effects of combining both GSK’212 and PD’991 in cells that are wild-type for BRAF and NRAS or an RB1-null BRAF mutant line. However, B-RAF and N-RAS mutant cells exhibited an enhanced response to the combinatorial agents as compared to single agents. Furthermore, we observed a dramatic increase in apoptosis in cell lines that are sensitive to combined GSK’212 and PD’991 treatment. Expression of Inhibitor-of-Apoptosis (IAP) protein, survivin, was associated with response. Finally, because it is important to understand how resistance develops, we have validated a novel E2F reporter system in vivo to quantitatively and temporally measure the efficacy of CDK4/6 inhibitors and possible re-activation of the pathway during acquired drug resistance.

Citation Format: Jessica L. Teh, Andrew Aplin. CDK4/6 as a therapeutic target in malignant melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3096. doi:10.1158/1538-7445.AM2015-3096
Abstract 3103: Cyclin E amplification predicts sensitivity of primary Uterine Serous Carcinoma (USC) cell lines to the cdk2 inhibitor CYC065
Authors: Cocco Emiliano , Bellone Stefania , Lopez Salvatore , Bonazzoli Elena , Predolini Federica , Black Jonathan D. , Santin Alessandro D.
Published: August 2015

We evaluated the in vitro effectiveness of the cdk2 inhibitor CYC065 on multiple primary chemotherapy-resistant USC cell lines with or without CCNE1 amplification.

CCNE1-amplified primary cell lines were significantly more sensitive than wild type USC cell lines to CYC065 in vitro (i.e., IC50: mean±STDV = 61.75±13.22 nM and 103.16± 21.9 nM for CCNE1-amplified USC-ARK-2 and USC-ARK-7 cell lines, respectively and 539.2±182.1 nM for the wild type USC-ARK-4 cell line, p = 0.0048). Consistently, low concentrations of CYC065 (i.e., 100 - 300 nM) caused a dose dependent arrest in the G1 phase of the cell cycle specifically in CCNE1-amplified primary USC cell lines. Importantly, CCNE1 knockdown in the USC-ARK-2 cell line resulted in a 9.29-fold increase in the CYC065 IC50 when compared to the MOCK-transfected USC-ARK-2 cell line (p = 0.021). Finally, when primary CCNE1-amplified USC cell lines also harboring ERBB2 amplification (50% of CCNE1-amplified USC cell lines) were incubated in vitro with the combination of CYC065 and Herceptin (a monoclonal antibody targeting the product of the ERBB2 gene, HER2/neu), an increased inhibitory effect was reported in the combination treatment when compared to Herceptin or CYC065 used as single agent (i.e.,% viable cells: mean±STDV = 71.4±0.85, 65.4±14.2, 42.2±2.1 for the Herceptin, the CYC065 and the combination treatment on USC-ARK-2, respectively; p = 0.014).

Together these findings identify CYC065 as a promising drug to be considered alone or in combination in the treatment of patients harboring CCNE1-amplified USC.

Citation Format: Emiliano Cocco, Stefania Bellone, Salvatore Lopez, Elena Bonazzoli, Federica Predolini, Jonathan D. Black, Alessandro D. Santin. Cyclin E amplification predicts sensitivity of primary Uterine Serous Carcinoma (USC) cell lines to the cdk2 inhibitor CYC065. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3103. doi:10.1158/1538-7445.AM2015-3103
Abstract 3106: Elucidating molecular mechanisms linking microRNA-206 loss to tumor progression and metastasis
Authors: Watt Kathleen D. , Truesdell Peter , Craig Andrew W.
Published: August 2015

Metastasis is the leading cause of cancer-related deaths, and this occurs when tumor cells invade basement membranes and blood vessels to colonize other tissues. Metastasis is facilitated by extracellular matrix (ECM)-degrading structures called invadopodia. Recently, several microRNAs (miRNAs) have been identified as tumor and metastasis suppressor genes. Loss of expression of miR-206 has been correlated with poor prognosis in gastric cancer, breast cancer, rhabdomyosarcoma and melanoma. A previous study by others has demonstrated that restoring miR-206 expression in human breast cancer cells was sufficient to block metastasis in tumor xenograft assays. However, the targets of miR-206 that cause metastasis in these cancer models have not been fully characterized. Here, we have tested the effects of miR-206 on invadopodia in metastatic cancer cells, and identified a novel target that may explain this effect. Transducer of Cdc42-mediated Actin assembly-1 (Toca-1) is an adaptor protein that promotes formation of invadopodia and metastasis in breast cancer models, and has a predicted miR-206 binding site within the 3′ UTR of Toca-1 transcripts. To test whether the high levels of Toca-1 expression that we have observed in metastatic cancer cell lines was due to loss of miR-206, we rescued miR-206 expression using transient, stable or inducible approaches in MDA-MB-231 breast cancer, A375 melanoma, and H1299 lung cancer cell lines. In each cell model, Toca-1 expression was reduced upon miR-206 expression at both the mRNA and protein levels, thus validating Toca-1 as a new target of miR-206. Further testing of the phenotypes induced by miR-206 rescue revealed dramatic defects in ECM degradation and cell invasion in breast cancer and melanoma cell models. In subcutaneous tumor xenograft assays, stable rescue of miR-206 in H1299 tumors led to defects in tumor growth compared to those expressing a scrambled control miRNA. In addition, miR-206 expression caused a dramatic reduction in the numbers of lung metastases. We are currently extending this to our melanoma models and inducible models to define changes in metastasis-related targets of miR-206 that are aberrantly expressed in metastatic cancers. In conclusion, our study demonstrates for the first time a link between miR-206 and suppression of invadopodia formation via Toca-1 silencing, which likely contributes to the metastasis suppressing activity of miR-206 in multiple cancer types.

Citation Format: Kathleen D. Watt, Peter Truesdell, Andrew W. Craig. Elucidating molecular mechanisms linking microRNA-206 loss to tumor progression and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3106. doi:10.1158/1538-7445.AM2015-3106
Abstract 311: High-throughput spheroid formation in a 384-well format using magnetic 3D bioprinting
Authors: Tseng Hubert , Gage Jacob A. , Haisler William L. , Souza Glauco R.
Published: August 2015

Technical limitations in the rapid and reproducible formation of multicellular tumor spheroids prevent their widespread use in high-throughput screening for cancer research and drug development. Existing systems for spheroid formation require lengthy processing times, and make simple tasks like media exchange and sample retention challenging. An ideal 3D cell culture system would print spheroids rapidly, while being easy to handle and process for high-throughput applications.

To address this unmet need, we use magnetic 3D bioprinting for high-throughput spheroid formation. Magnetic 3D bioprinting is based on the principle of magnetizing cells using a magnetic nanoparticle assembly. Once magnetized, these cells can be directed and aggregated using magnetic forces; specifically, magnetized cells can be aggregated into spheroids in 384-well plates. These spheroids can be cultured long-term, and, as they are magnetic, the spheroids can be held down or transferred out of the well, avoiding the technical issues of other 3D systems to retain samples. Moreover, the magnetic nanoparticles have no effect on cell behavior and do not interfere with fluorescence.

We demonstrated this method by printing spheroids of HepG2, HCT-116, A549, and PANC-1 cancer cells in 384-well plates. We printed spheroids rapidly (15 min) and reproducibly, with cell numbers as small as 100 cells. We assayed the spheroids by using their contraction over time as an endpoint, which we validated as a toxicity endpoint correlating with cell migration, proliferation, and viability. In running these experiments we also demonstrated that these spheroids are amenable to high-content testing, with multiple experiments being performed on the same spheroid to yield more data per test.

Thus, this study introduces magnetic 3D bioprinting for high-throughput spheroid formation, where multicellular tumor spheroids that represent native tumor environments can be rapidly and easily printed for cancer research and compound screening.

Citation Format: Hubert Tseng, Jacob A. Gage, William L. Haisler, Glauco R. Souza. High-throughput spheroid formation in a 384-well format using magnetic 3D bioprinting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 311. doi:10.1158/1538-7445.AM2015-311
Abstract 3117: Hsa-miR-140-5p inhibits colorectal cancer stem cell invasion and metastasis by suppressing Smad2 and autophagy
Authors: Zhai Haiyan , Fesler Andrew , Ju Jingfang
Published: August 2015

Colorectal cancer is one of the most common cancers and frequently metastasizes to the liver. TGF-β signaling has been shown to regulate cancer metastasis by promoting epithelial to mesenchymal transition and cancer stem cell (CSC) phenotypes. Smad2 is a key element downstream of TGF-β. In this study, we showed that hsa-hsa-miR-140-5p-5p directly targets Smad2 and overexpression of hsa-miR-140-5p in colorectal cancer cell lines decreases Smad2 expression level, which leads to decreased level of cell invasion and proliferation, as well as increasing cell cycle arrest. Ectopic expression of hsa-miR-140-5p in colorectal CSCs inhibited CSC growth and sphere formation in vitro by disrupting autophagy, an essential process for cancer cell survival and chemoresistance. We have systematically identified the targets of hsa-miR-140-5p involved in autophagy. Furthermore, overexpression of hsa-miR-140-5p in CSCs abolished the tumor formation as well as metastasis in vivo by using subcutaneous xenografts and tail vein injection of CSCs in NOD/SCID mice. In addition, there is a progressive loss of hsa-miR-140-5p expression from normal colorectal mucosa to primary tumor tissues, with further reduction in liver metastatic tissues, quantified by qRT-PCR. The functional and clinical significance of hsa-miR-140-5p suggests that it is a key regulator in colorectal cancer progression and metastasis. As a result, it may have potential as a novel therapeutic molecule to treat colorectal cancer.

Citation Format: Haiyan Zhai, Andrew Fesler, Jingfang Ju. Hsa-miR-140-5p inhibits colorectal cancer stem cell invasion and metastasis by suppressing Smad2 and autophagy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3117. doi:10.1158/1538-7445.AM2015-3117
Abstract 313: Examining the role of ABCA1 cholesterol transporter in ovarian cancer spheroids
Authors: Williams Rebekka , Russell Amanda , Bongers Angelika , Sagnella Sharon , Fife Christopher , Jessup Wendy , DeFazio Anna , Chenevix-Trench Georgia , Haber Michelle , Norris Murray , Henderson Michelle
Published: August 2015

Epithelial ovarian cancer (EOC) is a devastating disease which accounts for a large proportion of gynaecological cancer-related deaths. Poor survival is largely due to late diagnosis of the disease which typically presents with peritoneal dissemination. In order to achieve reduced tumor burden and improved survival, it is imperative to identify new prognostic markers and potential therapeutic targets. Recent work by our group identified high ATP- Binding Cassette A1 (ABCA1) transporter expression as being associated with poor outcome in EOC. ABCA1 has been widely studied as a cholesterol transporter, however there is little research investigating its potential roles in cancer biology. The aim of this work was to ascertain whether ABCA1 may promote aggressive characteristics in EOC. siRNA-mediated suppression of ABCA1 in EOC cell lines led to reduced colony forming ability, growth and migration by comparison with controls. By culturing EOC cell lines (Skov3, 27/87 and HEY) in a 3-dimensional spheroid model, we have shown that ABCA1 suppression significantly reduced overall spheroid volume (p = ≤0.0003, ANOVA with Dunnett's multiple comparison test). In Skov3 and 27/87 spheroids, reduced volume was associated with cell death; however in HEY spheroids, ABCA1 suppression significantly reduced cell numbers in the absence of detectable cell death (p = ≤0.0002, ANOVA with Dunnett's multiple comparison test). Detailed characterisation of spheroid architecture revealed an increased volume-to-cell ratio in those depleted of ABCA1, indicating a lower degree of compaction compared to controls. It was further observed that whilst HEY spheroids derived from control siRNA-transfected cells develop a prominent necrotic core this feature was dramatically impaired by ABCA1 suppression. Compaction and core necrosis are both well-documented processes in spheroid biology that are indicative of cellular contractility and a hypoxic tumour environment, both key characteristics of more aggressive, chemo-resistant and recurrent tumors. Collectively these data suggest that ABCA1 may be involved in regulating multiple processes associated with aggressive and invasive characteristics of EOC, highlighting ABCA1 as a potential therapeutic target.

Citation Format: Rebekka Williams, Amanda Russell, Angelika Bongers, Sharon Sagnella, Christopher Fife, Wendy Jessup, Anna DeFazio, Georgia Chenevix-Trench, Michelle Haber, Murray Norris, Michelle Henderson. Examining the role of ABCA1 cholesterol transporter in ovarian cancer spheroids. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 313. doi:10.1158/1538-7445.AM2015-313
Abstract 3130: Discovery of tumor suppressor microRNA using smoking-related lung carcinogenesis cell model
Authors: Sung Jae Sook , Lee Jong Won , Kim Yeul Hong
Published: August 2015

Cigarette smoking is an important risk factor for lung cancer and induces DNA hypermethylation. Hypermethylation can silence tumor suppressor miRNAs. However, it is less known regarding the role of miRNA methylation in lung carcinogenesis, especially smoking-related lung cancer. We are now to discover the smoking-induced altered tumor suppressor miRNA in human lung carcinogenesis cell model. First, we examined miRNA expression profiles using Affymetrix GeneChip miRNA4.0 in human lung carcinogenesis cell model; NHBE, BEAS2B, 1799, 1198, 1170I cells (PNAS. 89:6693-97. 1992, Cancer Biol Ther. 5:665-73. 2006). And we have validated candidate tumor suppressor miRNAs by TaqMan real time PCR. We first discovered 6 candidate tumor suppressor miRNAs (miR-4448, miR-3180-3p, miR-584-5p, miR-3658, miR-8064, miR-371b) and we confirmed known decreased expression of several miRNAs in lung carcinogenesis cell model. Next, we tested methylation of candidate tumor suppressor miRNAs promoter CpG site by pyrosequencing. Increase of methylation was observed for 4 of the 6 candidate tumor suppressor miRNAs in human lung carcinogenesis cell model. Furthermore, methylation of miR-584-5p, miR-3658, miR-8064, miR-371b were greater in 1198 and 1170I cells exposed to cigarette smoking condensate (CSC). In the further study, we will confirm the role of tumor suppressor miRNA in smoking-related lung carcinogenesis by functional study. These finding suggest that tumor suppressor miRNAs are miRNA silenced by epigenetic mechanisms during smoking-induced lung carcinogenesis. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A1A2062146) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI13C1611).

Citation Format: Jae Sook Sung, Jong Won Lee, Yeul Hong Kim. Discovery of tumor suppressor microRNA using smoking-related lung carcinogenesis cell model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3130. doi:10.1158/1538-7445.AM2015-3130