bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1900: MiR-222 accelerates the progression of inflammatory breast cancer by targeting tumor suppressor gene p27kip1 (CDKN1B)
Authors: Ogony Joshua Were , Hayes Erin , Knapp Jennifer , Lewis-Wambi Joan
Published: July 2016

MicroRNAs (miRNAs) are small non-coding RNAs about 18-22 nucleotides in length, whose main role is to regulate gene expression by altering mRNA stability and translation. MicroRNAs have been shown to play a role in many types of cancer, including breast cancer by targeting tumor suppressor genes such as p53, p27, PTEN, BRCA1, BRCA2 and many others. The goal of this study was to investigate the role of miRNAs in inflammatory breast cancer (IBC). IBC is the most aggressive and metastatic form of breast cancer, that is characterized by very rapid progression, poor prognosis, with a 5 year survival outcome of 30-40%. The mechanism that underlies the rapid progression of IBC is currently under intense investigation, and novel approaches are needed to better understand this disease. To understand the role that microRNAs might play in IBC, we conducted next generation sequencing of the miRNAs from two IBC cell line, SUM149 which is a triple negative IBC (ER-, PR-, HER2-), and SUM190 which is HER2 overexpressing, but ER and PR negative (ER-, PR-, HER2+). We found that 463 miRNAs were differentially expressed in the two cell lines, with 243 miRNAs being overexpressed in SUM149 compared to SUM190, while 220 miRNAs were overexpressed in SUM190. Notably, our qPCR analysis confirmed the raw read counts, showing that miR-155, miR-221, and miR-222 were overexpressed in SUM149 as compared to SUM190 cells by 15, 10, and 3 fold respectively. Furthermore, the inhibition of miR-222 in SUM149 resulted in significant reduction in cell proliferation, tumorsphere formation, and migration in these cells as compared to SUM190 cells. Our flow cytometry data showed that there was cell cycle arrest at the G1 phase in SUM149 cells due to miR-222 inhibition, while SUM190 cells showed little or no change. Additionally, Western blot and immunofluorescence analyses showed that the protein levels of p27, which is one of the targets of miR-222, significantly increased in SUM149 cells, but not in SUM190 cells when miR-222 inhibitor was used in these cells. These results suggest that miR-222 may accelerate the progression of some IBCs by targeting tumor suppressor genes, resulting in rapid cell proliferation, and may be a potential therapeutic target for triple negative inflammatory breast cancers.

Citation Format: Joshua Were Ogony, Erin Hayes, Jennifer Knapp, Joan Lewis-Wambi. MiR-222 accelerates the progression of inflammatory breast cancer by targeting tumor suppressor gene p27kip1 (CDKN1B). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1900.
Abstract 1938: The identification of p53 target genes and noncoding RNAs through the combined analysis of RNA-seq and ChIP-seq data
Authors: Idogawa Masashi , Ohashi Tomoko , Sasaki Yasushi , Tokino Takashi
Published: July 2016

p53 is inactivated in approximately half of human cancers. p53 execute various functions by modulating transcriptional regulation. To identify direct p53 transcriptional targets including non-coding RNAs, we performed gene expression analysis with next-generation sequencing (RNA-seq) in osteosarcoma U2OS cells treated with Nutlin-3a which activates endogenous p53. We identified 261 genes increased more than 4-fold and 373 long non-coding RNAs (lncRNAs) increased more than 2-fold. Next, we analyzed the data of chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) and then searched for p53 binding sites in the whole human genome. p53 binding sites were located in the neighborhood of 126/261 (48.3%) genes including 28 known target genes and 122/373 (32.7%) lncRNAs including 7 lncRNAs we already identified in previous study. Recent reports have revealed that lncRNAs play an important role in various biological and pathological processes such as development, differentiation, stemness and carcinogenesis. Although all functions of lncRNAs have not been elucidated, many lncRNAs are involved in transcriptional regulation like transcriptional factors. Therefore, we performed a gene network analysis of cancer transcriptome including lncRNAs using public RNA-seq data of cancer tissues. As a result, we identified several hub lncRNAs which regulate the transcription of many downstream genes. Our results suggest that p53 and lncRNAs comprise a complex transcriptional network for various biological functions and tumor suppression.

Citation Format: Masashi Idogawa, Tomoko Ohashi, Yasushi Sasaki, Takashi Tokino. The identification of p53 target genes and noncoding RNAs through the combined analysis of RNA-seq and ChIP-seq data. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1938.
Abstract 1942: Change in serum microRNA expression during neoadjuvant chemoradiation for rectal cancer
Authors: Lim Stephanie H. , De Souza Paul , Chua Wei , Ng Weng , Harris Benjamin , Cowley Mark J. , Spring Kevin
Published: July 2016

Introduction

MicroRNAs (miRNA) expression may change with treatment and influence responses in locally advanced rectal cancer (LARC) making them potentially useful predictive and prognostic biomarkers. We investigated 112 miRNAs in serum during chemoradiation for LARC.

Methods

Twenty-one patients with LARC treated with neoadjuvant chemoradiation were prospectively recruited for the study. Serum was collected at three time-points: 1) at diagnosis (baseline), 2) at three weeks into treatment and 3) at completion of chemoradiation (pre-surgery). Serum was also collected from 10 healthy controls. RNA extraction was performed using the Norgen™ total RNA purification kit. Reverse transcription and pre-amplification were performed as per the Taqman™ OpenArray MicroRNA Panels manufacturer's instructions. miRNA array qPCR was performed on 112 miRNA targets using the QuantStudio™ 12K platform. Differentially expressed miRNAs were identified using the delta-delta-Ct method, using the endogenous U6 snRNA as the control. Analysis was performed in R, using paired t-statistics, and the Benjamini-Hochberg False Discovery Rate for multiple hypothesis testing adjustment, with a threshold q<0.05 for significant differential expression. Enriched KEGG pathways were identified using DIANA, based on verified gene targets of each miRNA from Tarbase.

Results

We identified 12 miRNAs that were significantly differentially expressed between patients and matched controls. The strongest differences were observed between patient samples at baseline and completion of chemoradiation, where eight miRNAs decreased in expression: hsa-miR-101, hsa-miR-130a, hsa-miR-130b, hsa-miR-223, hsa-miR-27a, hsa-miR-628-5p, hsa-miR-630 and hsa-miR-720, all with fold change > 3-fold. These genes are known to target key rectal cancer genes such as SMAD4, BCL2, MSH2 and TGFBR2. An additional three miRNAs changed significantly between baseline and week 3: hsa-miR-135b*, hsa-miR-375 and hsa-miR-629. All except hsa-miR-135b* became less abundant.

Conclusions

Ten differentially expressed miRNAs appear downregulated during chemoradiation in LARC. These miRNAs have been implicated in cell proliferation, the epithelial-mesenchymal transition and radiation resistance. Further work will be undertaken to understand the functional implications of these changes.

Citation Format: Stephanie H. Lim, Paul De Souza, Wei Chua, Weng Ng, Benjamin Harris, Mark J. Cowley, Kevin Spring. Change in serum microRNA expression during neoadjuvant chemoradiation for rectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1942.
Abstract 1979: Comprehensive analysis of the dynamics of aberrant WNT signaling on global gene expression and higher order nuclear structure in human cells
Authors: Brown Markus , Wangsa Darawalee , Hu Yue , Ried Thomas
Published: July 2016

The organization of the genome in the nucleus is complex and dynamic. Features of the nuclear architecture, including the spatial arrangement of genomic sequences, the structure of chromatin, and the accessibility of regulatory DNA elements modulate nuclear processes such as gene transcription DNA replication, DNA repair, RNA processing, and mRNA transport. However, the interplay between nuclear architecture and gene expression is poorly understood. We propose to elucidate the impact of silencing the prominent transcription factor, TCF7L2, the major effector of the WNT signaling pathway, on the transcriptional equilibrium and nuclear architecture of the nucleus in human colorectal cancer cells. Our multidisciplinary approach combines targeted silencing of TCF7L2, 3D-FISH, RNA-seq, and Hi-C chromosome conformation capture, in parallel with mathematical modeling to delineate structure/function relationships in the interphase nucleus. This approach will elucidate the intricacies of a dynamic cellular signaling pathway in three-dimensional space.

Citation Format: Markus Brown, Darawalee Wangsa, Yue Hu, Thomas Ried. Comprehensive analysis of the dynamics of aberrant WNT signaling on global gene expression and higher order nuclear structure in human cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1979.
Abstract 1986: CEBPD promotes stemness in the pathogenesis of gliomas
Authors: Wang Shao-Ming , Ko Chiung-Yuan , Wang Ju-Ming
Published: July 2016

Glioblastoma multiforme (GBM) is the most common and lethal type of brain tumor with a median survival of 12-15 months despite optimal therapy. Nowadays, an increasing number of studies has indicated the connection between malignancy and stemness. The molecular mechanisms controlling the stem cell-like properties and tumorigenic potential of glioblastoma stem cells (GSCs) remain less investigated. CCAAT/Enhancer binding protein delta (CEBPD) is a C/EBP family of transcription factors. In this study, CEBPD was highly expressed in GBM specimens and GBM patients with low levels of CEBPD show a significantly better overall survival. The transcription factor OCT4 is known to play an important role in maintaining stem cell self-renewal within the central nervous system (CNS) and this activity is present in gliomas as well. Further, our data showed that CEBPD contributes to stem-like characteristics and promoted cancer cells toward stem cell traits through direct activation of OCT4 gene expression. In conclusion, this study highlights the involvement of CEBPD in enchaining stem-cell property in GBM.

Citation Format: Shao-Ming Wang, Chiung-Yuan Ko, Ju-Ming Wang. CEBPD promotes stemness in the pathogenesis of gliomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1986.
Abstract 2009: Role of FOXO1 in oncogenic program of B cell precursor acute lymphoblastic leukemia (BCP-ALL)
Authors: Wang Fan , Ushmorov Alexey , Wirth Thomas
Published: July 2016

Although treatment of B cell precursor acute lymphoblastic leukemia (BCP-ALL) in children has shown eminent progress over the past decades, about 15% of patients can still not be cured, however. Therefore, new therapeutic strategies are warranted.

FOXO1 together with other transcription factors including EBF1, PAX5 and TCF3 plays a central role in differentiation of pre-B-cells. In cooperation with PAX5 and other B-cell transcription factors, FOXO1 induces expression of SYK, BLNK, RAG1, RAG2, AICDA and IL7RA genes. Of note, SYK and IL7RA signaling contribute to the oncogenic program of BCP-ALL, which utilize pre-B cell survival and proliferation program.

In this study, we demonstrate that FOXO1 is highly expressed in most of the BCP-ALL cell lines. By using immunofluorescence assays we show that FOXO1 is localized in the nucleus of BCP-ALL cells. Overexpression of constitutively active AKT1 (inactivating FOXO1) negatively regulates the growth of BCP-ALL cells. Inhibition of FOXO1 by shRNA results in cell death in the RS4;11 BCP-ALL cell line. BCP-ALL cells are more sensitive to growth arrest and apoptosis induced by a small molecular FOXO1 inhibitor AS1842856 than other classical Hodgkin lymphoma and non-Hodgkin lymphoma cell lines. Gene expression profiling of BCP-ALL cells after treatment with the FOXO1 inhibitor for 24h shows the significantly down-regulation of FOXO target genes including VPREB3, IL1B, KLHL24 and BNIP3L. Interestingly, at the same time, some FOXO targets like NOXA, RUNX2 and PRDM1 were found upregulated, indicating a complex effect of the FOXO1 inhibitor.

We conclude that FOXO1 expression is critical for the BCP-ALL oncogenic program and its targeting represents a novel therapeutic approach.

Citation Format: Fan Wang, Alexey Ushmorov, Thomas Wirth. Role of FOXO1 in oncogenic program of B cell precursor acute lymphoblastic leukemia (BCP-ALL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2009.
Abstract 2016: Paired box 2 promotes progression of endometrial cancer via cell cycle pathway
Authors: Wang Jieyu , Feng Weiwei , Yu Yinhua , Wong Kwong-Kwok , Jia Nan , Lyu Tianjiao
Published: July 2016

Human paired box 2 (PAX2) plays a key role in cell fate, early patterning and organogenesis. To investigate the function of PAX2 on the biological behavior of endometrial cancer and the regulation mechanism, stable knocking-down and over-expression PAX2 endometrial cancer cell lines were established. CCK-8 and transwell assays were applied to determine proliferation, invasion and migration ability. Cell cycle distribution was analyzed by flow cytometry. Nude mice were used for in vivo experiments and Affymetrix GeneChip® human Exon 1.0 ST arrays was used to screen the downstream target genes of PAX2. The upstream regulation mechanism of PAX2 was explored by dual luciferase reporter gene assay and bioinformatics analysis.Results showed that,no matter in vivo or in vitro,PAX2 significantly enhanced tumorigenicity and invasiveness of endometrial cancer cells, and promoted liver metastasis in vivo. In addition, PAX2 influenced the expression of cyclin-dependent kinase 1(CDK1) and YWHAZ, which play pivotal roles in cell cycle pathway. Knocking down CDK1 in PAX2 over-expressing cells attenuated cell viability.A myeloid zinc finger gene-1(MZF-1)binding site was found in a negative regulation region of PAX2 upstream and down-regulation of MZF-1 reduced the transcriptional activity of PAX2, as well as its protein level.Together, PAX2, through influencing the activity of CDK1 and YWHAZ, promotes proliferation,invasion and progression of endometrial cancer and can be regulated by MZF-1. PAX2 may be a target therapeutic site for endometrial cancer.

Citation Format: Jieyu Wang, Weiwei Feng, Yinhua Yu, Kwong-Kwok Wong, Nan Jia, Tianjiao Lyu. Paired box 2 promotes progression of endometrial cancer via cell cycle pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2016.
Abstract 2056: Development of quaternary amine linkers for ADCs: Application to auristatin E and tubulysin
Authors: Burke Patrick J. , Hamilton Joseph Z. , Pires Thomas A. , Setter Jocelyn R. , Hunter Joshua H. , Cochran Julia H. , Toki Brian E. , Senter Peter D. , Lyon Robert P. , Jeffrey Scott C.
Published: July 2016

The recent clinical success of antibody-drug conjugates (ADCs) has spawned an increased effort to identify new technologies, and the development of new drug-linker chemistry is vital to expand the scope of conjugatable payloads. The tertiary amine functional group is a common structural motif present in many bioactive compounds, including antimitotics of the auristatin and tubulysin classes. Traditionally, conjugation of tertiary amines required drug derivatization or modification to remove an N-alkyl group, thus creating a readily conjugatable secondary amine. However, identifying appropriate modifications that do not compromise the activity of the drug is frequently time consuming and often unsuccessful. To eliminate the need for such structural modifications, we sought a method for stable conjugation and facile release through the tertiary amine functional group by creating linkers with a quaternary amine point of attachment. To validate the linker strategy, quaternary amine-based cleavable linkers bearing auristatin E were synthesized and evaluated as ADCs. The conjugates were stable in rodent plasma, and were potent and immunologically specific both in vitro and in vivo in a Hodgkin lymphoma xenograft model. A second application of this technology has been demonstrated with tubulysins, another class of potent antimitotics containing a tertiary amine at the N-terminus. A cleavable quaternary amine linker containing a tubulysin analog was synthesized and ADCs were prepared and evaluated. The tubulysin conjugates were potent and immunologically specific across a panel of cancer cell lines, including multiple MDR-positive lines. Furthermore, the tubulysin conjugate displayed ‘bystander activity’ in an in vitro co-culture assay. The quaternary amine linkers represent an advance in linker technology and will enable the evaluation of drug classes previously inaccessible as ADCs.

Citation Format: Patrick J. Burke, Joseph Z. Hamilton, Thomas A. Pires, Jocelyn R. Setter, Joshua H. Hunter, Julia H. Cochran, Brian E. Toki, Peter D. Senter, Robert P. Lyon, Scott C. Jeffrey. Development of quaternary amine linkers for ADCs: Application to auristatin E and tubulysin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2056.
Abstract 2059: Identification of novel paclitaxel sensitivity genes via lentiviral CRISPR/Cas9 screening in haploid human cells
Authors: Hunt Jessica R. , Lawo Steffen , Walter David , Nett Isabelle , Yarker Joanne , Cross Benedict C S , Moore Jonathan D.
Published: July 2016

Some cancers lack clearly druggable driver mutations and might also have too few neo-antigen generating mutations to provoke a tumor-specific immune response, even in the presence of antibodies targeting immune checkpoint mechanisms. Synthetic lethal/non-oncogene addiction represents an alternative approach to the therapy of such cancers, as exemplified by the recent approval of olaparib.

RNA interference has identified several putative synthetic lethal targets, but few, if any, of these have have withstood vigorous validation. The recent application of RNA-guided gene editing to mammalian cells offers new prospects for target identification. The complete ablation of gene expression, which this technology permits allows the identification of targets that have no phenotype when partially depleted by RNA interference.

Horizon has recently established a capability for CRISPR-Cas9 KO screening. Screens are performed by transducing a pool of cells with a complex lentiviral library. Next generation sequencing is then used to identify which sgRNAs are accumulating or being depleted from the population. As there are multiple sgRNAs per gene, we can rank genes for their likelihood of having a phenotype under the conditions used in the screen. Our proof-of-principle small molecule resistance and sensitivity screens using both whole-genome libraries and custom subset libraries have successfully identified previously published hits and also yielded novel hits.

Horizon has also developed a fully haploid cell line (eHAP), which has the potential to provide additional clarity in genetic screening. To evaluate the power of CRISPR-Cas9 KO screening in haploid cells, we have used our screening platform to identify genes required for survival in low doses of paclitaxel. Whole-genome screening using the GeCKOv2 library identified three kinesins amongst the most prominent of the hits whose depletion sensitised cells to this microtubule stabilising agent. Intriguingly, two of these kinesins, KIF2A and KIF2C are thought to destabilise mitotic microtubules, providing an attractive explanation for the synthetic lethality.

We will also present results from a second genome-wide CRISPR KO screen in haploid cells to identify genes required for survival under glucose starvation. Prominent hits included components of the electron transport chain and the tuberous sclerosis complex.

Citation Format: Jessica R. Hunt, Steffen Lawo, David Walter, Isabelle Nett, Joanne Yarker, Benedict C S Cross, Jonathan D. Moore. Identification of novel paclitaxel sensitivity genes via lentiviral CRISPR/Cas9 screening in haploid human cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2059.
Abstract 2066: Albumin-binding doxorubicin prodrug selectively activated by radiation-induced apoptotic tumor cells
Authors: Chung Seung Woo , Kim Kwangmeyung , Kim Seong Who , Kim In-San , Kim Sang Yoon , Byun Youngro
Published: July 2016

Despite the well-recognized potent anticancer effect of doxorubicin, the clinical use of doxorubicin is limited due to its dose-dependent toxicity. In this study, a novel drug delivery technology fundamentally different from the conventional tumor-targeting approaches was employed to improve the therapeutic index of doxorubicin. The prepared doxorubicin prodrug, EMC-DEVD-S-DOX, has two distinct features for effective tumor targeting: EPR effect-mediated passive targeting of tumor and extended plasma half-life by in situ albumin binding, and radiation-induced apoptosis targeting. The prodrug comprises of two important functional molecules: first, a maleimide group that allows in situ binding of the prodrug to the circulating albumin after intravenous administration; second, a DEVD motif that plays a crucial role in the activation of the prodrug through caspase-3-mediated cleavage in the apoptotic tumor cells that are exposed to radiation. As a result, EMC-DEVD-S-DOX showed a prolonged plasma half-life with selective accumulation within the tumor tissue, and was only activated and released free doxorubicin when combined with radiotherapy, thereby showing excellent synergistic effect. Our suggested drug delivery strategy completely relies on the anatomical feature of tumor vasculatures and the upregulated caspase-3 after exposure to radiation. In this reason, the genomic variations of tumor cells are unlikely to influence the efficacy of the prodrug. Considering that the conventional active tumor targeting approach is seriously challenged by the intratumor heterogeneity, EMC-DEVD-S-DOX could be an outstanding alternative for an effective cancer treatment.

Citation Format: Seung Woo Chung, Kwangmeyung Kim, Seong Who Kim, In-San Kim, Sang Yoon Kim, Youngro Byun. Albumin-binding doxorubicin prodrug selectively activated by radiation-induced apoptotic tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2066.
Abstract 2067: Attacking prostate cancer with a prodrug-doped cellular Trojan horse
Authors: Brennen Nathaniel , Levy Oren , Han Edward , Rosen David Marc , Musabeyezu Juliet , Safaee Helia , Ranganath Sudhir , Ngai Jessica , Heinelt Martina , Billett Sandrine , Bhowmick Neil , Denmeade Samuel , Karp Jeffrey , Isaacs John
Published: July 2016

Prostate cancer is the second leading cause of cancer-related deaths in American men. Despite considerable advances in prostate cancer research, there is a major need for a systemic delivery platform that efficiently targets anti-cancer drugs to sites of disseminated prostate cancer while minimizing host toxicity. Human mesenchymal stem cells (MSCs) are excellent candidates for cell-based drug delivery since they display tropism towards cancer sites and clinical studies have demonstrated that hundreds of millions of allogeneic human MSCs can be safely administered intravenously without adverse effects in a variety of pathological settings. Furthermore, we have previously documented that MSCs can be detected in radical prostatectomy tissue from men with prostate cancer. In this proof-of-concept study, human MSCs were loaded with poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) encapsulating the macromolecule G114, a thapsigargin-based prostate specific antigen (PSA)-activated prodrug. G114-loaded MPs were successfully internalized by MSCs without impacting MSC viability, followed by sustained release of G114 as an intact prodrug from loaded cells. Moreover, G114 released from G114 MP-loaded MSCs is selectively toxic to the PSA-secreting prostate cancer cell line, LNCaP. Finally, G114 MP-loaded MSCs inhibited tumor growth when co-inoculated with CWR22 prostate cancer xenografts, suggesting that cell-based delivery of G114 does not compromise potency of the prodrug in vitro or in vivo. We envision that this MSC-based platform may be developed into an effective, systemic ‘Trojan Horse’ therapy for the targeted delivery of therapeutic agents to sites of metastatic prostate cancer.

Citation Format: Nathaniel Brennen, Oren Levy, Edward Han, David Marc Rosen, Juliet Musabeyezu, Helia Safaee, Sudhir Ranganath, Jessica Ngai, Martina Heinelt, Sandrine Billett, Neil Bhowmick, Samuel Denmeade, Jeffrey Karp, John Isaacs. Attacking prostate cancer with a prodrug-doped cellular Trojan horse. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2067.
Abstract 2077: Carbonate prodrugs derived from triterpenoids with high cytotoxic activity
Authors: Dzubak Petr , Burianova Renata , Michalova Martina , Liskova Barbora , Urban Milan , Sarek Jan , Galandakova Adela , Ulrichova Jitka , Hajduch Marian
Published: July 2016

Number of active anticancer compounds failed in the drug development due to unfavorable pharmacological properties. Design of pro-drugs is plausible approach to improve drug-like properties of biologically active compounds. Betulinic acid is a well-known representative of triterpenes, which possess a moderate cytotoxic activity and high selectivity towards the tumor cells. However, its pharmacological properties, particularly solubility and bioavailability, are poor. In order to improve pharmacology of betulinic acid derivatives, we have synthesized and tested activities of above 40 carbonate based prodrugs. We have developed methods for activation of “pro-drug” derivatives of betulinic acid by primary rat hepatocytes. Cytotoxicity of pro-drugs and activated metabolites was measured on the sensitive tumor cell line CCRF-CEM (human T-lymphoblastic leukemia). We have identified 21 prodrugs >10 times more cytotoxic to CCRF-CEM cells after pre-incubation with rat hepatocytes. The best candidate pro-drug showed >100 fold increase of cytotoxicity due to metabolic activation and it was cytotoxic to cancer cells in sub-micromolar concentrations. Our concept and predicted mechanisms of pro-drug activation were verified by HPLC-MS/MS where the pro-drugs and corresponding active compounds were identified and quantified. For the most active derivatives we have tested ADME characteristics and in vivo pharmacokinetics which showed significantly improved drug-like properties. Analysis of anti-cancer activities of the pro-drugs is currently being performed. This work was supported by grants LF_2015_031 and by the National Sustainability Program (LO1304).

Citation Format: Petr Dzubak, Renata Burianova, Martina Michalova, Barbora Liskova, Milan Urban, Jan Sarek, Adela Galandakova, Jitka Ulrichova, Marian Hajduch. Carbonate prodrugs derived from triterpenoids with high cytotoxic activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2077.
Abstract 209: The effect of heme influx on initiation and tumorigenesis of NSCLC
Authors: Sohoni Sagar , Alam Md Maksudul , Vidal Chantal , Hooda Jagmohan , Zhang Li
Published: July 2016

Heme is a central molecule for mitochondrial function and for all processes involved in oxygen utilization. Heme serves as a prosthetic group or as a cofactor for a number of oxidative phosphorylation enzymes and other oxygen-utilizing hemoproteins. Heme also directly regulates the synthesis, translocation and assembly of these enzyme complexes. Most, if not all, human cells can synthesize and uptake heme from the circulation. A number of epidemiological studies have shown that high heme intake is associated with increased risk of cancer, including lung cancer. Recent studies carried out in our lab showed intensified mitochondrial respiration and increased levels of heme and hemoproteins in non-small-cell lung cancer (NSCLC) cells. Together, these experimental and epidemiological studies strongly suggest that heme is an “oncometabolite.” To assess the status of heme metabolism in cancer cells, we performed a series of experiments in NSCLC cells and compared the results with an immortalized normal lung cell line, HBEC30KT. We used Zinc protoporphyrin, an analogue of heme to measure the level of heme uptake. We found that heme uptake as well as heme synthesis are significantly elevated in all NSCLC cells compared to HBEC. The rate of heme degradation was also measured in these cell lines. Previously, our lab demonstrated that lowering intracellular heme levels selectively decreases oxygen consumption in NSCLC cells and inhibits cell migration and colony formation. Experiments are underway to test ways to alter intracellular heme availability and characterize their effects on NSCLC tumor growth and metastasis.

Citation Format: Sagar Sohoni, Md Maksudul Alam, Chantal Vidal, Jagmohan Hooda, Li Zhang. The effect of heme influx on initiation and tumorigenesis of NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 209.
Abstract 2153: ATP7B is a promising therapeutic target for uterine leiomyosarcoma
Authors: Kakuda Mamoru
Published: July 2016

Objective

Resistance to platinum drugs remains a significant problem in uterine leiomyosarcoma (LMS). We investigated the role of ATP7B in the resistance to platinum drugs in LMS using both in vitro and in vivo models.

Methods

The expression of the typical platinum transporters (MDR1, MRP2, ATP7A, and ATP7B) was examined in LMS cell lines using Western blotting analysis. ATP7A expression was investigated by immunohistochemistry (IHC) using clinical samples of LMS. IC50 values to cisplatin were measured in SK-LMS cells, SK-LMS-ATP7B-suppressed cell line (SK-LMS-7B cells), which permanently transfected PRS ATP7B shRNA vector. We established xenografted mice by inoculating SK-LMS cells and SK-LMS-7B cells, and examined in vivo platinum sensitivity with cisplatin for both tumors. This study was approved by the Institutional Review Board and the Ethics Committee of the Osaka University Hospital (approval #10302, approved on March 11, 2011).

Results

The expression of ATP7B was identified in the SK-LMS cells. ATP7B expression was identified in 70% (14/20) of the LMS clinical samples using IHC. The IC50-values to cisplatin improved from 17 mM to 4.3 mM after the suppression of ATP7B in SK-LMS-7B cells. A significant anti-tumor effect of cisplatin was observed in SK-LMS-7B xenografted mice than in SK-LMS xenografted mice. We also identified CuSO4 as an preferentially inhibitor of ATP7B in vitro.

Conclusion

ATP7B is associated with platinum resistance. CuSO4 acting as an inhibitor of ATP7B can be a therapeutic target for LMS.

Citation Format: Mamoru Kakuda. ATP7B is a promising therapeutic target for uterine leiomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2153.
Abstract 2155: Acquired tamoxifen resistance sensitises breast cancer cells to bisphosphonates
Authors: Lymperatou Dionysia , Smith Christopher , Jiang Wen Guo , Evans Bronwen , Hiscox Stephen
Published: July 2016

Acquisition of resistance to endocrine therapies is a major limiting factor to their clinical effectiveness, resulting in disease relapse and poor prognosis. This may be due in part to the observations that acquired resistance to agents such as tamoxifen is accompanied by a gain in aggressive cellular features likely to promote disease spread and survival at metastatic sites. Bisphosphonates (BPs) are potent antiresorptive drugs used to treat osteoporosis and bone metastasis. Recent evidence also suggests that these agents may possess anti-tumour properties independently of their action on osteoclasts in a range of different tumours including breast cancers resulting in suppression of proliferation and migration and an induction of apoptosis. Here we have explored the potential of the bisphosphonate, zoledronic acid (ZOL) to suppress the aggressive phenotype of acquired endocrine-resistant breast cancer models versus their endocrine-sensitive counterparts.

ER+, endocrine-sensitive MCF7 cells and their highly-proliferative, ER+, tamoxifen-resistant (‘TamR’) counterparts were exposed to increasing concentrations of ZOL (1-160μM) for 3 days and changes in growth determined by MTT assay and cell counting. Immunohistochemical analysis of Ki67 confirmed cell growth effects whilst cell cycle analysis was performed using FACS and propidium iodide. Changes in signalling pathways were investigated by Western blotting.

The proliferative capacity of TamR cells was greatly suppressed by ZOL in a dose dependent manner in contrast to MCF7, where the effect was minimal (IC50 ± SD: 30 ± 4 μM (TamR) versus 150 ± 15 μM (MCF7); p<0.05). This was confirmed with Ki67 analysis which showed a reduction in Ki67 positivity of 40% (TamR) versus 25% (MCF7); p<0.05. ZOL treatment of TamR cells resulted in a significant loss of cells in G1 phase and an accumulation of the cells in S phase. ZOL treatment of TamR cells also resulted in loss of AKT activity, whilst AKT was unaffected in MCF7 cells.

Our data suggests that acquisition of resistance to tamoxifen confers a sensitivity to BPs where treatment of resistant cells with agents such as zoledronic acid results in suppression of proliferation and modulation of AKT signalling. These data suggest that BPs may exert direct anti-tumour effects on breast cancers which have relapsed on endocrine treatment.

Citation Format: Dionysia Lymperatou, Christopher Smith, Wen Guo Jiang, Bronwen Evans, Stephen Hiscox. Acquired tamoxifen resistance sensitises breast cancer cells to bisphosphonates. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2155.
Abstract 216: The effect of UCP2 upregulation on cellular redox status and mitochondrial respiration
Authors: Sreedhar Annapoorna , Zhao Yunfeng
Published: July 2016

Uncoupling protein 2 (UCP2) is a mitochondrial protein present in the inner mitochondrial membrane. They belong to the family of anion mitochondrial carriers. UCP2 are thought to have natural antioxidant effect by attenuating the generation of superoxides.

Since cancer cells exhibit elevated oxidative stress, they can effectively adapt to this situation by upregulating UCP2. As a matter of fact, UCP2 is overexpressed in many forms of cancer including prostate, breast, lung and head & neck. Not surprising, our early studies demonstrate that knockout of UCP2 suppresses skin tumor formation in a multistage skin carcinogenesis model.

The purpose of this study was to detect the impact of UCP2's upregulation on mitochondrial redox status and mitochondrial respiration, using the well characterized tumor promotion model, JB6p+ cells. Our results have demonstrated that UCP2 overexpression enhanced protein expression and activity levels of manganese superoxide dismutase (MnSOD), a mitochondrial-resident enzyme that governs the egression of superoxides. To assess the impact of MnSOD upregulation on mitochondrial redox status, levels of superoxides and hydrogen peroxides were detected. Our results indicated that MnSOD upregulation maintained steady flow of hydrogen peroxides originating from mitochondria. We further examined the effect of UCP2 upregulation-induced MnSOD expression on mitochondrial respiration and glycolytic rate. In agreement with our data, cells overexpressing UCP2 had significantly higher oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). In conclusion, our data suggests that UCP2 promotes carcinogenesis via decreasing superoxide generation whereas promoting hydrogen peroxide production via elevating MnSOD. Collectively, our results support the hypothesis that enhanced UCP2 expression sustains metabolic switch in cancer cells.

Citation Format: Annapoorna Sreedhar, Yunfeng Zhao. The effect of UCP2 upregulation on cellular redox status and mitochondrial respiration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 216.
Abstract 217: Mitochondrial reprogramming regulated cancer pathway in triple negative breast cancer
Authors: Park Jun H. , Vithayathil Sajna , Wu Danli , Putluri Vasanta , Sung Pi-Lin , Tsouko Efrosini , Bhat Vadiraja B. , Coarfa Cristian , Frigo Daniel E. , Lewis Michael T. , Sreekumar Arun , Yotnda Patricia , Creighton Chad J. , Putluri Nagireddy , Wong Lee-Jun C. , Kaipparettu Benny A.
Published: July 2016

Compared to other subtypes of tumors, triple negative breast cancers (TN BCa) currently suffer from limited knowledge on its etiology and treatment options. Transmitochondrial cybrids (cybrid) and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in TN BCa. Analysis of cybrids and established BCa cell lines showed that metastatic TN BCa maintain high levels of ATP through fatty acid β-oxidation and activate Src oncoprotein by its autophosphorylation. Inhibition and induction of β-oxidation including the shRNA mediated knockdown strategies, and analysis of patient derived xenograft (PDX) models confirmed the role of mitochondrial β-oxidation in Src activation and metastasis. Analysis of BCa clinical data further reaffirmed the role of mitochondrial β-oxidation in Src regulation and their significance in BCa metastasis. This study is innovative in showing the mitochondrial reprogramming mediated regulation of a major cancer pathway by its post-translation modification.

Citation Format: Jun H. Park, Sajna Vithayathil, Danli Wu, Vasanta Putluri, Pi-Lin Sung, Efrosini Tsouko, Vadiraja B. Bhat, Cristian Coarfa, Daniel E. Frigo, Michael T. Lewis, Arun Sreekumar, Patricia Yotnda, Chad J. Creighton, Nagireddy Putluri, Lee-Jun C. Wong, Benny A. Kaipparettu. Mitochondrial reprogramming regulated cancer pathway in triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 217.
Abstract 2174: A high-throughput, high-content assay for the discovery of new inhibitors of DNA double-strand break repair
Authors: Surovtseva Yulia , Jairam Vikram , Sundaram Ranjini , Bindra Ranjit , Herzon Seth
Published: July 2016

DNA repair pathways are being intensively investigated as targets for chemotherapeutic interventions. Despite the immense interest in this area, few whole-cell screens for the discovery of DNA repair inhibitors have been described. We have developed an automated, whole-cell, high-throughput assay for the unbiased discovery of inhibitors of the non-homologous end joining (NHEJ) and homologous recombination HR) repair pathways, the two primary pathways that ameliorate DNA double-strand breaks. Our assay enables indirect measurement of DNA repair activity by monitoring the kinetics of γH2AX and 53BP1 foci resolution after treatment with ionizing radiation (IR) and candidate DNA damage response (DDR) inhibitor. We also have developed a unique platform of secondary assays to rank-order the potency of lead compounds and gain insights into their mechanism of action. In addition to hit validation and rigorous exclusion of false-positive hits, our follow-up platform includes orthogonal assays for measuring mutagenic NHEJ and HR repair activity, as well as high-throughput assays for quantitative analysis of cell viability and cell cycle. Notably, both our primary and secondary assays are fully-automated and may be applied to screen large libraries of compounds.

We validated our high-throughput assay using known DDR and DNA damage checkpoint inhibitors, and applied this screen toward the evaluation of 2,366 structurally-diverse small molecules with known bioactivity. Vorinostat and quercetin, compounds known to possess DDR inhibitory activity, were identified in the screen, supporting the sensitivity of our approach. Notably, the screen also lead to the identification of cardiac glycosides, natural products in clinical use for the treatment of heart failure and atrial arrhythmia, as potent inhibitors of DNA double-strand break repair. Many studies have noted the anticancer properties of the cardiac glycosides, but the basis of this activity has remained a “black box”. Our data suggest an explanation for this long-standing unsolved problem, and argue that this activity should be taken into consideration when evaluating other clinical applications of these agents. Because these compounds are already approved for use in humans, we believe they are excellent candidates for repurposing as chemo and radiosensitizers.

Citation Format: Yulia Surovtseva, Vikram Jairam, Ranjini Sundaram, Ranjit Bindra, Seth Herzon. A high-throughput, high-content assay for the discovery of new inhibitors of DNA double-strand break repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2174.
Abstract 2227: Positive correlation between the gamma-H2AX and PD-L1 expressions in lung squamous cell carcinoma
Authors: Osoegawa Atsushi , Hashimoto Takafumi , Takumi Yohei , Abe Miyuki , Hiraishi Hitomi , Suehiro Shuji , Miyawaki Michiyo , Sugio Kenji
Published: July 2016

Lung cancer is a leading cause of cancer death worldwide. Recently, molecular targeting therapy has been developed and it has shown promising results against advanced lung cancer. Among them, lung squamous cell carcinoma has fewer treatment options because it is not driven by oncogenic mutations, but by alterations in tumor suppressor genes and subsequent chromosomal instability. Programmed death-ligand 1 (PD-L1) is one of the immune checkpoint molecules that is expressed on the surface of tumor cells, where it inhibits activities of cytotoxic T cells. Recent studies revealed that the PD-1/PD-L1 blockade could improve the overall survival in lung squamous cell carcinoma, potentially because it carries high mutation burdens and neoantigens which could be targeted by cytotoxic T cells. We hypothesized that DNA damage could accumulate in tumors with high mutation burdens, thereby inducing the PD-L1 expression on tumor cells, sensitizing them to anti-PD-1 therapy. An immunohistochemical analysis of 41 consecutive lung squamous cell carcinoma cases, which received surgery at our institution between April 2013 and March 2014, revealed that a high PD-L1 expression was observed in 15 patients (37%) that was associated related with a poor recurrence-free survival (p = 0.028). The PD-L1 expression level was also positively associated with the γH2AX expression (a direct marker for DNA damage) (p = 0.02). The γH2AX expression in tumor cell nuclei was confirmed by immunofluorescent staining. It forms foci in tumor cell nuclei, indicating the incidence of DNA double strand breaks. Our findings demonstrate for the first time that the nuclear γH2AX expression in lung squamous cell carcinoma is positively associated with the PD-L1 expression. Further studies are warranted to investigate whether γH2AX could be a biomarker for PD-1 targeting therapy and whether combination therapy with PD-1 targeting therapy and a DNA-damaging agent has synergistic effects.

Citation Format: Atsushi Osoegawa, Takafumi Hashimoto, Yohei Takumi, Miyuki Abe, Hitomi Hiraishi, Shuji Suehiro, Michiyo Miyawaki, Kenji Sugio. Positive correlation between the gamma-H2AX and PD-L1 expressions in lung squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2227.
Abstract 2270: A BRCA1 deficient, NFκB driven immune signal predicts good outcome in triple negative breast cancer
Authors: Buckley Niamh E. , Haddock Paula , De Matos Simoes Ricardo , Parkes Eileen , Irwin Gareth , Emmert-Streib Frank , McQuaid Stephen , Kennedy Richard D. , Mullan Paul B.
Published: July 2016

Triple negative (TNBCs) and the closely related Basal-like (BLBCs) breast cancers are a loosely defined collection of cancers with poor clinical outcomes. Both show strong similarities with BRCA1-mutant breast cancers and BRCA1 dysfunction, or ‘BRCAness’, is observed in a large proportion of sporadic BLBCs. BRCA1 expression and function has been shown in vitro to modulate responses to radiation and chemotherapy. Exploitation of this knowledge in the treatment of BRCA1-mutant patients has had varying degrees of success. This reflects the significant problem of accurately detecting those patients with BRCA1 dysfunction. Moreover, not all BRCA1 mutations/loss of function result in the same histology/pathology or indeed have similar effects in modulating therapeutic responses. Given the poor clinical outcomes and lack of targeted therapy for these subtypes, a better understanding of the biology underlying these diseases is required in order to develop novel therapeutic strategies.

We have discovered a consistent NFκB hyperactivity associated with BRCA1 dysfunction as a consequence of increased Reactive Oxygen Species (ROS). This biology is found in a subset of BRCA1-mutant and triple negative breast cancer cases and confers good outcome. The increased NFκB signalling results in an anti-tumour microenvironment which may allow CD8+ cytotoxic T cells to suppress tumour progression. However, tumours lacking this NFκB-driven biology have a more tumour-promoting environment and so are associated with poorer prognosis. Tumour-derived gene expression data and cell line models imply that these tumours may benefit from alternative treatment strategies such as reprogramming the microenvironment and targeting the IGF and AR signalling pathways.

Citation Format: Niamh E. Buckley, Paula Haddock, Ricardo De Matos Simoes, Eileen Parkes, Gareth Irwin, Frank Emmert-Streib, Stephen McQuaid, Richard D. Kennedy, Paul B. Mullan. A BRCA1 deficient, NFκB driven immune signal predicts good outcome in triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2270.