bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5367: Elucidating the factors determining P53 binding and transcriptomic response to DNA damage
Authors: Sundaresan Varsha , Li Ying , DiCiaccio Benedetto , Lin Victor T. , Higaki Adrian A. Acuna , Pelletier Nicole F. , Cheung Jessica T. , Zhou Lei
Published: August 2018

Mutations in tp53 are thought to be one of the most common mechanisms by which cancer cells evade tumor suppression. Yet the exact mechanism of tumor suppression by P53 remains to be fully understood. The transcription factor P53 is activated in response to oncogenic stresses and exerts distinct anti-proliferative functions based on the stressor and cell type. Although numerous mammalian ChIP-Seq studies have identified thousands of P53 binding sites, the functionality of these binding sites remains to be established.

To study how P53 binding following DNA damage differs between cell types, we performed comparisons between P53 ChIP-Seq data from Drosophila embryos at different developmental stages and a Drosophila cell line (Kc167). Differential expression analysis using RNA-Seq indicated that at an early stem cell-like developmental stage of Drosophila embryos there is P53-dependent induction of pro-apoptotic genes in response to irradiation but not in the later differentiated stages. We aim to establish functionally significant P53 binding sites by combining ChIP-Seq and RNA-Seq data from Drosophila embryos as well as Kc167. The functional significance of these binding sites will be identified by CRISPR-Cas9–mediated genome editing. Preliminary genome-wide motif analysis studies also exhibited that homotypic clustering of P53 consensus motifs is associated with P53 binding. We will also perform comparison studies of DNA damage-induced P53 binding in humans, mice and flies to identify analogous patterns.

Preliminary studies revealed that a majority of the P53 binding sites are located at the repetitive regions of the Drosophila genome, especially the Long terminal repeats (LTRs) and Long interspersed nuclear elements (LINEs) as seen previously in mammals by other groups including Botcheva et al and Chang et al. P53 has been known to regulate the expression of retrotransposons and maintain genetic stability by keeping the number of repeats in control. We further aim to see if the functions carried out by P53 differ between repetitive vs. non-repetitive binding regions in Drosophila.

This comparative genomics study will help identify the functionally significant P53 binding sites thus further our understanding of tumor suppression by P53. The knowledge obtained from this study will also be valuable in devising novel strategies to restore P53 function in cancers.

Citation Format: Varsha Sundaresan, Ying Li, Benedetto DiCiaccio, Victor T. Lin, Adrian A. Acuna Higaki, Nicole F. Pelletier, Jessica T. Cheung, Lei Zhou. Elucidating the factors determining P53 binding and transcriptomic response to DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5367.
Abstract 5374: Uterine sarcomas: A preliminary study for characterization of a specific molecular signature for Brazilian patients
Authors: Costa Leonardo T. , Torrezan Giovana T. , Paula Claudia A. , Neto Glauco B. , Baracat Edmund C. , Carraro Dirce M. , Carvalho Kátia C.
Published: August 2018

Background: Uterine sarcomas are the most common gynecological sarcomas, comprising around 90% of all of the cases. These tumors can arise either from the endometrial stroma or from the muscle itself. They are genetically heterogeneous tumors and have no specific molecular and radiological diagnosis, or treatment.

Histologically, uterine sarcomas are classified according to their cell of origin and are divided into: carcinosarcomas (CCS), leiomyosarcomas (LMS), endometrial stromal sarcoma (ESS) and adenosarcomas (ADS). There are many factors that can be attributed to uterine sarcomas etiopathogeny such as ethnic, hormonal and hereditary factors. However, few studies were performed searching for specific molecular signature in these tumors, especially concerning a singular population. Objective: To assess the oncogenic profile of Brazilian patients with different histological types of uterine sarcomas. Methods: Until now, we performed the Next Generation Sequencing for 13 samples of uterine sarcomas (comprising: 5 CCS, 3 LMS, 4 SEE and 1 ADS) using the Ion Proton System (ThermoFisher) with the Comprehensive Cancer Panel (containing 409 genes related to carcinogenesis). Results: The genetic sequencing profile analysis showed 140 specific mutations in our samples. Among them, the most common were TP53 (58%), JAK3 (33%), NF1 (33%), KMT2D (33%), MTOR (25%) and PIK3CA (25%). Some findings corroborate the literature on uterine sarcomas, with an exception of the KMT2D gene, that despite of being known and described in other tumors, including the ones of the genital tract, such as vulva, ovary and endometrium, has never been described in uterine sarcomas Conclusion: We found the most frequent mutations in 6 genes in our samples. Only one mutation had not been described before for uterine sarcomas (KMT2D). These are preliminary results, and further analyzes are ongoing.Key words: Uterine Sarcomas, Carcinosarcomas, Next Generation Sequencing

Citation Format: Leonardo T. Costa, Giovana T. Torrezan, Claudia A. Paula, Glauco B. Neto, Edmund C. Baracat, Dirce M. Carraro, Kátia C. Carvalho. Uterine sarcomas: A preliminary study for characterization of a specific molecular signature for Brazilian patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5374.
Abstract 538: ASPM promotes prostate cancer tumorigenesis by bolstering cancer stemness through Wnt-Dvl-3-b-catenin signaling
Authors: Tsai Kelvin K. , Pai Vincent C. , Hsu Chung-Chi , Chan Tze-Sian
Published: August 2018

Metastasis is the major cause of cancer mortality in androgen-independent prostate cancer (PCA) and understanding it is crucial to improving the outcome of patients. Recent evidence suggests that activation of Wnt signaling in cancer stem cells (CSCs) contributes to cancer progression in malignant tumors. Here we report that a novel Wnt activator ASPM (Abnormal spindle-like microcephaly associated) maintains the prostate CSC subpopulation through augmenting the Wnt-β-catenin signaling in PCA. Functional studies showed that downregulation of ASPM expression attenuated the proliferation and invasive of PCA cells and reduced the number of prostate CSCs and inhibited cancer stemness and tumorigenesis. Mechanistically, ASPM interacts with dishevelled-3 (Dvl-3) and inhibits its proteasome-dependent degradation, thereby increasing the protein stability of Dvl-3 and enabling the β-catenin transcriptional activity in PCA cells. The clinical significance of the above findings was credentialed by the profound up-regulation of ASPM in metastatic PCA and the strong correlation of cytoplasmic ASPM expression with poor metastasis-free survival of patients with resected PCA. Collectively, our data points to ASPM as a novel oncoprotein and an essential regulator of cancer stemness in PCA, which has important clinical and therapeutic significance (supported by Ministry of Science and Technology grants MOST 104-2314-B-400-022 and MOST 105-2314-B-400-018 and National Health Research Institutes NHRIs grant CA-106-PP-09 to K.K.T).

Citation Format: Kelvin K. Tsai, Vincent C. Pai, Chung-Chi Hsu, Tze-Sian Chan. ASPM promotes prostate cancer tumorigenesis by bolstering cancer stemness through Wnt-Dvl-3-b-catenin signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 538.
Abstract 5405: Multiplex profiling of miRNA biomarkers for discovery and verification using the FirePlex® platform
Authors: Atabakhsh Elnaz , Kelley Ivona , Tackett Michael , Diwan Izzuddin , Tejada Gensis , Rafferty Conor , Pregibon Daniel
Published: August 2018

To address the needs for microRNA (miRNA) biomarker discovery and verification, we developed the FirePlex® miRNA assay. This assay enables the detection of 5-400 miRNA targets per sample in 96-well format, with readout on standard flow cytometers and analysis with an included bioinformatics software package. The FirePlex miRNA assay combines particle-based multiplexing, using patented FirePlex® hydrogel particles, with single step RT-PCR signal amplification using universal primers. Thus, the FirePlex miRNA assay leverages PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target-specific qPCR. The miRNA assay can reliably detect as few as 1000 miRNA copies per sample with a linear dynamic range of ~5 logs, and without the need of prior RNA purification, making the assay ideally suited for profiling in serum, plasma, exosomes, urine, and directly from FFPE tissues. Furthermore, the ability to multiplex targets in each well eliminates the need to split valuable samples into multiple reactions. Results from the FirePlex miRNA assay are displayed and interpreted using the integrated FirePlex Analysis Workbench, which allows visualization, normalization, and export of experimental data with only a few mouse clicks. To aid biomarker discovery studies, we offer pre-designed panels of 400 miRNAs based on a research-curated list of miRNAs published to be found in circulating biofluids. For studies focused on specific research areas of interest, we offer pre-designed focus panels for Oncology, Cardiology, Neurology, Immunology, and Organ Toxicity. These carefully curated panels include hemolysis markers to assess sample quality, as well as critical normalization factors. Here we present the data from several studies investigating circulating miRNA profiles, as well as miRNA profiles obtained directly from FFPE tissues, using the FirePlex miRNA Assay Panels. Together, this novel combination of bioinformatics tools and multiplexed, high-sensitivity assays enables rapid discovery and verification of miRNA biomarker signatures from biofluid samples.

Citation Format: Elnaz Atabakhsh, Ivona Kelley, Michael Tackett, Izzuddin Diwan, Gensis Tejada, Conor Rafferty, Daniel Pregibon. Multiplex profiling of miRNA biomarkers for discovery and verification using the FirePlex® platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5405.
Abstract 5411: Micro-RNA expression in cemento-ossifying fibroma
Authors: Pereira Thais S. , Guimarães André Luiz S. , Gomes Carolina C. , Gomez Ricardo S. , Diniz Marina G.
Published: August 2018

Cemento-ossifying fibroma (COF) is a benign fibro-osseous neoplasm of uncertain pathogenesis and its treatment is associated with significant morbidity. MicroRNAs (miRNA) are small non-coding RNAs that regulate gene expression and may represent therapeutic targets. The purpose of the study was to generate a comprehensive miRNA profile of COF compared to normal bone. Additionally, the most relevant pathways and target genes of differentially expressed miRNA were investigated by in silico analysis. Nine COF and ten normal bone samples were included in the study. miRNA profiling was carried out by using TaqMan® OpenArray® Human MicroRNA panel containing 754 validated human miRNAs. We identified the most relevant miRNAs target genes through the leader gene approach, using STRING and Cytoscape software. Pathways enrichment analysis was performed using DIANA-miRPath. Eleven miRNAs were downregulated (hsa-miR-95-3p, hsa-miR-141-3p, hsa-miR-205-5p, hsa-miR-223-3p, hsa-miR-31-5p, hsa-miR-944, hsa-miR-200b-3p, hsa-miR-135b-5p, hsa-miR-31-3p, hsa-miR-223-5p, hsa-miR-200c-3p) and five were upregulated (hsa-miR-181a-5p, hsa-miR-181c-5p, hsa-miR-149-5p, hsa-miR-138-5p, hsa-miR-199a-3p) in COF compared to normal bone. Eighteen common target genes were predicted, and the leader genes approach identified the following genes involved in human COF: EZH2, XIAP, MET and TGFBR1. According to the biology of bone and COF, the most relevant Kegg-pathways revealed by enrichment analysis were Proteoglycans in cancer, miRNAs in cancer, Pathways in cancer, p53, PI3K-Akt, FoxO, and TGF-beta signalling pathways, which were previously found to be differentially regulated in bone neoplasms, odontogenic tumours and osteogenesis. Dysregulation of miRNAs occurs in COF and EZH2, XIAP, MET and TGFBR1 are potential targets for functional analysis validation. Supported by: CNPq, CAPES and FAPEMIG (Brazil).

Citation Format: Thais S. Pereira, André Luiz S. Guimarães, Carolina C. Gomes, Ricardo S. Gomez, Marina G. Diniz. Micro-RNA expression in cemento-ossifying fibroma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5411.
Abstract 5416: Isolation of the PAX8 transcriptional complex to identify novel therapeutic vulnerabilities for ovarian cancer
Authors: Chaves-Moreira Daniele , Mitchell Marilyn , Sidoli Simone , Garcia Benjamin , Drapkin Ronny
Published: August 2018

A central problem in the treatment of ovarian cancer remains the heterogeneity among ovarian tumors. DNA and RNA sequencing studies have demonstrated both intertumoral and intratumoral genetic variation. Despite efforts to elucidate common signaling pathways among various ovarian cancer subtypes, few have led to meaningful patient stratification or to truly individualized targeted molecular therapies. Our proposed set of experiments is a radical departure from the conventional approach to treating ovarian cancer. PAX8, a transcription factor that identifies nearly all high-grade serous ovarian cancers (HGSOCs), is also the master regulator of fallopian tube development. Building off observations pointing to the fallopian tube epithelium as a major site of origin for HGSOCs, we propose targeting the fallopian tube developmental program as the basis for new therapies. This possibility is supported by the observation that knockdown of PAX8 leads to apoptosis in ovarian cancer cells. We hypothesize that blocking the ability of PAX8 to influence its gene targets, either by interrupting PAX8 protein-protein interactions or by inhibiting the products of PAX8-driven signaling, has the potential to eliminate the primary growth stimulus for high-grade serous tumors. In order to achieve that goal, we have identified candidate PAX8-interacting partners using gel filtration chromatography and tandem-affinity immunopurification coupled with mass spectrometry analyses. Interactions have been validated by Western blot, immunofluorescence, and proximity ligation assay. We used ovarian cancer (KURAMOCHI, OVSAHO and OVCAR4) and fallopian tube (FT189, FT194 and FT246) cell lines to determine whether PAX8 protein partners changed between benign and malignant cells. Our preliminary results suggest that PAX8 interacts with other transcription factors such as TEAD4 and components of chromatin-remodeling complexes (RUVBL1, RUVBL2 and CHD4). Many of these factors are known to mediate the effects of oncogenes such as YAP, β-catenin and c-Myc. Further studies will be done in order to decipher the functional contribution of each candidate PAX8-interacting protein. Our ultimate goal is to determine how PAX8 regulates expression of its target genes and how PAX8-interacting proteins influence that function. Inhibition of certain protein-protein interactions may provide novel avenues to abrogate PAX8 function in ovarian cancer cells.

Citation Format: Daniele Chaves-Moreira, Marilyn Mitchell, Simone Sidoli, Benjamin Garcia, Ronny Drapkin. Isolation of the PAX8 transcriptional complex to identify novel therapeutic vulnerabilities for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5416.
Abstract 5418: GATA3 zinc-finger mutation induces transcriptional reprogramming in breast cancer through gain and loss of function
Authors: Takaku Motoki , Grimm Sara A. , Roberts John D. , Chrysovergis Kaliopi , Myers Page , Perou Charles M. , Wade Paul A.
Published: August 2018

Recent large-scale breast cancer genomic profiling identified frequent mutations in a pioneer transcription factor, GATA3. The expression of GATA3 is a prominent marker of luminal breast tumors, and loss of GATA3 expression is associated with aggressive tumor phenotypes. In addition, GATA3 mutations are frequently found in these luminal tumors. However, the clinical and molecular outcomes of GATA3 mutations are poorly understood. Our novel classification scheme of GATA3 mutations defines distinct clinical features of breast tumors with mutations in the second GATA3 zinc-finger (ZnFn2): frequent observation in luminal B subtype and worse prognosis than other GATA3 mutant cases. To dissect the molecular impacts of these ZnFn2 mutations, we generated a GATA3 mutant breast cancer cell clone by CRISPR-Cas9 gene editing. An engineered ZnFn2 mutant cell line manifested increased tumor growth as xenografts and more aggressive phenotypes in vitro. The ZnFn2 mutation led to loss of GATA3 binding and decreased expression at a subset of genes, including progesterone receptor (PR). The mutant cells also exhibited gain of GATA3 binding at other loci, correlated with increased expression of mesenchymal marker genes. Decreased expression of PR and impaired response to progesterone were crucial for cancer-promoting functions in GATA3 ZnFn2 mutant cells. Downregulation of PR and its downstream genes was also observed in the clinical gene expression data. These results illuminate tumor-promoting functions of GATA3 ZnFn2 mutations in breast cancer.

Citation Format: Motoki Takaku, Sara A. Grimm, John D. Roberts, Kaliopi Chrysovergis, Page Myers, Charles M. Perou, Paul A. Wade. GATA3 zinc-finger mutation induces transcriptional reprogramming in breast cancer through gain and loss of function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5418.
Abstract 5424: Improved anticancer activity of clotam as a copper-complex
Authors: Sankpal Umesh T. , Patel Rafid , Arechiga Bianca , Hunter Abigail , Chhabra Jaya , Brown Deondra T. , Gurung Raj K. , Holder Alvin A. , Basha Riyaz M.
Published: August 2018

Clotam (tolfenamic acid or TA) is a nonsteroidal anti-inflammatory drug used to treat migraine headaches. Research from our laboratory and other groups has demonstrated that TA also acts as an anticancer agent in several adult and pediatric cancer models. It is well established that the anticancer activity of TA is partly mediated by its ability to downregulate the specificity protein (Sp) family of transcription factors and their downstream targets, notably the inhibitor of apoptosis protein family member survivin. Both Sp1 and survivin are upregulated in cancer cells and are associated with poor prognosis, making them ideal therapeutic targets. It was recently suggested that the therapeutic potential of TA could be enhanced by forming a complex with copper(II). In this study we synthesized a copper(II) complex of TA (Cu-TA) and tested its anticancer activity using cell lines representing various cancers such as breast, prostate, pancreatic, brain, colon, neuroblastoma, medulloblastoma, and sarcoma. The compound was synthesized and its purity was assessed by UV-visible spectrophotometry. Purified compound (>98%) was dissolved in DMSO and its antiproliferative activity was determined using the CellTiter-Glo cell viability assay kit. In our initial screening various cancer cells were treated with increasing concentration of Cu-TA or TA, and cell viability was assessed at 24 and 48h post-treatment. The effect of Cu-TA on expression of Sp1 and survivin was determined by Western blot analysis of lysates prepared using cells treated with Cu-TA or TA for 48h. We also studied the effect of Cu-TA on cell cycle and apoptosis using medulloblastoma cell lines. Both Cu-TA and TA decreased cell viability in a dose-dependent manner; however, Cu-TA consistently had lower IC50 values in all the cell lines tested, suggesting its higher efficacy compared to TA. In line with the viability results, Western blot analysis of survivin expression demonstrated that Cu-TA was more effective in inhibiting its expression compared to TA. Also, treatment of medulloblastoma cells with Cu-TA resulted in cell cycle arrest in G0/G1 phase accompanied by an increased apoptosis as determined by annexin-V staining. This preliminary screening demonstrates the potential of Cu-TA as an effective anticancer agent with the ability to target survivin. Further studies to delineate the mechanism of action of Cu-TA are under way.

Citation Format: Umesh T. Sankpal, Rafid Patel, Bianca Arechiga, Abigail Hunter, Jaya Chhabra, Deondra T. Brown, Raj K. Gurung, Alvin A. Holder, Riyaz M. Basha. Improved anticancer activity of clotam as a copper-complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5424.
Abstract 5425: Investigating the effects of p53 activation and HDAC inhibition on genes associated with poor prognosis prostate cancer
Authors: Gregg Gemma M. , Falcone Fiammetta , Lees Andrea , Quinn Gerard , Gallagher Peter , Kennedy Richard , Mills Ian I. , McDade Simon S.
Published: August 2018

Background

Prostate cancer (PCa) is now the most commonly diagnosed malignancy in men worldwide. SOC combines radiotherapy with ADT, however recurrence rate remains unacceptably high and many men present with late stage aggressive and metastatic disease, with poor prognosis and limited treatment options.

Despite tumor suppressor p53 being mutated in approximately 50% of human tumors, it has been reported that p53 alteration occurs in only 12% of primary prostate tumors. Emerging data links epigenetic-mediated gene silencing as a major differentiator between aggressive and indolent disease that is associated with up-regulation of key cell cycle regulator FOXM1, which is known to be suppressed by p53. HDAC inhibitors (HDACi) are an emerging class of epigenetic modifying anti-cancer agents, which we hypothesises will enhance p53 activity, cell death and alter patterns of gene expression to targeting p53 dependent and independent vulnerabilities in PCa.

Methods

We used basally p53 deficient/proficient PCa models (PC3/LNCaP) and novel C4-2B and LNCap p53 CRISPR-Cas9 knockout models to investigate the combination effects of Nutlin 3A or radiation (IR) with class 1 HDACi Entinostat by Annexin V/PI Flow Cytometry and Western Blot. Transcriptional effects were evaluated by RT-PCR and RNA-seq analysis. TCGA mutational and RNA-seq data were used to link p53 mutation with altered gene expression and outcome.

Results

We have demonstrated that there is a synergistic induction of cell death when we combine N3A/IR with Entinostat that is p53-dependent. In addition, we see that p53 acts as a barrier to genes associated with poor prognosis including FOXM1 which is phenocopied by single-agent Entinostat independent of WT-p53. This suppression may potentially be downstream of p21 via the DREAM complex. Importantly p53 mutation in TCGA primary PCa patients is associated with poor outcome and elevated FOXM1 expression.

Conclusion and Future Work

Results to date suggest that Entinostat enhances p53 dependent cell death but also has p53 dependent and independent mechanisms to suppress genes associated with poor prognosis PCa. The mechanism looks to be dependent of p21 and may involve DREAM complex.

Citation Format: Gemma M. Gregg, Fiammetta Falcone, Andrea Lees, Gerard Quinn, Peter Gallagher, Richard Kennedy, Ian I. Mills, Simon S. McDade. Investigating the effects of p53 activation and HDAC inhibition on genes associated with poor prognosis prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5425.
Abstract 5427: Inhibition of hypoxia-inducible factor 1 (HIF-1) transcriptional activity and cell mobility via G9a/GLP-mediated methylation of HIF-1α
Authors: Bao Lei , Chen Yan , Lai Hsien-Tsung , Chiang Cheng-Ming , Semenza Gregg L. , Wang Yingfei , Luo Weibo
Published: August 2018

Hypoxia-inducible factor 1 (HIF-1) is a main regulator of cellular response to hypoxia and its transcriptional activity is crucial for cancer cell migration and invasion. Post-translational modifications of the α subunit of HIF-1 (HIF-1α) play dominant roles in regulating its function. In this study, we demonstrated that the lysine methyltransferases G9a and GLP catalyzed mono- and di-methylation of HIF-1α at lysine (K) 674 in vitro and in vivo. Functionally, K674 methylation suppressed HIF-1 transcriptional activity as well as expression of the HIF-1 target genes including PTGS1, NDNF, SLC6A3, and Linc01132 in human glioblastoma U251MG cells. Moreover, mutation of K674 of HIF-1α significantly enhanced U251MG cell migration under hypoxia. Importantly, we found that G9a level was downregulated by hypoxia in glioblastoma, which was negatively correlated with PTGS1 expression and survival rate in glioblastoma patients. These findings reveal a novel naturally existing negative feedback mechanism that maintains high activity of HIF-1 and cell mobility in human glioblastoma.

Citation Format: Lei Bao, Yan Chen, Hsien-Tsung Lai, Cheng-Ming Chiang, Gregg L. Semenza, Yingfei Wang, Weibo Luo. Inhibition of hypoxia-inducible factor 1 (HIF-1) transcriptional activity and cell mobility via G9a/GLP-mediated methylation of HIF-1α [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5427.
Abstract 5428: Effect of amentoflavone on Nrf2 signaling in HaCaT cells
Authors: Wahyudi Lilik Duwi , Jeong Jiwon , Yang Heejung , Kim Jung-Hwan
Published: August 2018

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key mediator of cell response to oxidative stress and xenobiotics. Multiple lines of evidence suggest that Nrf2 activation protects against many diseases, aging, inflammation, and cancer. To date, Nrf2 activators from natural products have been widely studied. In this study we investigated the potential effect of Amentoflavone (AFN), a biflavonoid in many natural plants, on Nrf2 signaling in human keratinocytes cells (HaCaT). As results, AFN significantly increased ARE-luciferase activity and nuclear accumulation of Nrf2. Furthermore, a Nrf2 target protein, NQO-1, was significantly increased by AFN in dose- and time-dependent manner. To verify the Nrf2 signaling mechanism by AFN, firstly we measured reactive oxygen species (ROS) using DCFDA because mild intracellular ROS could stimulate Nrf2 activation. Surprisingly AFN triggered the production of ROS in 1 h and AFN-induced Nrf2 was inhibited by N-acetyl cysteine (NAC). Thus, we studied the ROS-related signaling on Nrf2 by measuring the activation of Akt and mitogen-activated protein (MAP) kinase family such as extracellular signal-regulated kinase (ERK) and p38. As results, pharmacology inhibitor of PI3K/Akt (LY294002) or p38 (SB 203580), but not ERK (U0126), abrogated AFN-activated Nrf2. Furthermore, silencing p38 using p38siRNA or inhibition using its pharmacology inhibitor decreased phosphorylation of Akt and attenuated Nrf2 activation. We also confirmed that AFN stabilized Nrf2 by inhibiting the ubiquitination. Taken together, AFN activates Nrf2 through ROS-mediated phosphorylation of p38 and followed by phosphorylation of Akt.

Citation Format: Lilik Duwi Wahyudi, Jiwon Jeong, Heejung Yang, Jung-Hwan Kim. Effect of amentoflavone on Nrf2 signaling in HaCaT cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5428.
Abstract 5433: XRCC5 promotes tumor growth and progression via PDK-1 signaling and predicts poor prognosis in melanoma
Authors: Qin Ge , Liu Tianze , Li Wenbin , Li Yixin , Zhang Changlin , Long Qian , Shi Dingbo , Chen Miao , Huang Shuting , Deng Wuguo
Published: August 2018

Melanoma is one of the most malignant and aggressive cancers with high cancer-related deaths. Patients with melanoma are often diagnosed at an advanced stage that has developed local or even remote metastasis due to its inapparent symptoms at early stages. Melanoma is often insensitive to chemotherapies, and most patients that respond well to targeted therapies or immunotherapies at first will develop resistance within several months. Therefore, it is of urgency to better understand the mechanisms of melanoma tumorigenesis, and to discover novel molecular targets for its effective treatment. In this study, we identified XRCC5 as a potential therapeutic target by siRNA library screening in A375 melanoma cells. We found that knockdown of XRCC5 inhibited cell growth, migration and invasion, and induced apoptosis in melanoma cells. Conversely,overexpression of XRCC5 significantly promoted the growth, migration and invasion of melanoma cells. We next analyzed the global transcriptome in A375 cells with XRCC5 knockdown, and identified PDK-1 as a candidate gene regulated by XRCC5. Further investigation revealed that XRCC5 cooperated with HIF-1α to bind at the promoter of PDK-1, and enhanced the expression of PDK-1. Consistently, overexpression of PDK-1 rescued the cell growth inhibition caused by XRCC5 knockdown. Our clinical data showed that the expression of XRCC5 and PDK-1 were positively correlated and elevated in melanoma samples, and high expression of XRCC5 predicted poor prognosis in melanoma patients. Collectively, our study discovered that XRCC5 bound to the promoter of PDK-1 to activate its expression, which promoted melanoma growth and progression. Our results indicated that the XRCC5/PDK-1 pathway could be a potential therapeutic target for melanoma.

Citation Format: Ge Qin, Tianze Liu, Wenbin Li, Yixin Li, Changlin Zhang, Qian Long, Dingbo Shi, Miao Chen, Shuting Huang, Wuguo Deng. XRCC5 promotes tumor growth and progression via PDK-1 signaling and predicts poor prognosis in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5433.
Abstract 5444: APIO-EE-9 is a novel Aurora A and B antagonist that suppresses esophageal cancer growth in a PDX mouse model
Authors: jin Guoguo
Published: August 2018

Esophageal cancer (EC) is one of the most aggressive malignancies of the upper aerodigestive tract. Over the past three decades, with advances in surgical techniques and treatment, the prognosis of esophageal cancer has only slowly improved. Thus identifying novel molecular targets and developing therapeutic agents are critical. Aurora kinases play a crucial role in mitosis and selective inhibitors might provide an effective therapeutic treatment for cancer. However, the role of Aurora kinases in EC is still inadequately studied. Here, we identified a novel compound, referred to as APIO- EE-9, which inhibits growth and colony formation and induces apoptosis of esophageal cancer cells. Using computer modeling, we found that APIO-EE-9 interacted with both Aurora A and B in the ATP-binding pocket. APIO-EE-9 inhibited both Aurora A and B kinase activities in a dose-dependent manner. Treatment with APIO-EE-9 substantially reduced the downstream Aurora kinase phosphorylation of histone H3 (Ser10), resulting in formation of multiple nuclei and centrosomes. Additionally, esophageal cancer cells expressing shAurora A or shAurora B kinase exhibited a dramatic reduction in proliferation and colony formation. Injection of these cells as xenografts in mice reduced tumor formation compared to wildtype cells. Importantly, APIO-EE-9 significantly decreased the size of esophageal patient-derived xenograft (PDX) tumors implanted in SCID mice. These results demonstrated that APIO-EE-9 is a specific Aurora kinase inhibitor that could be developed as a therapeutic agent against esophageal cancer.

Citation Format: Guoguo jin. APIO-EE-9 is a novel Aurora A and B antagonist that suppresses esophageal cancer growth in a PDX mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5444.
Abstract 5452: Pimozide suppresses the growth of brain tumor by targeting oncogenic pathways
Authors: Kaushik Itishree , Ranjan Alok , Schwettmann Blake , Srivastava Sanjay
Published: August 2018

Brain tumor is considered as one of the most aggressive and incurable form of cancer. Majority of the brain tumors have a median survival rate of only 12%. Even though advanced treatments such as surgical removal, chemotherapy and radiotherapy are available, brain tumor persists to be lethal. Obstacles associated with the current treatment options are: recurrence, development of resistance and inability to cross blood brain barrier (BBB). BBB restricts majority of drugs to reach the brain thus resulting in an ineffective treatment. Pimozide (PMZ) is an antipsychotic drug used for the treatment of schizophrenia and chronic psychosis. In this study, pimozide has shown significant reduction in the viability of U-87MG, U-251MG, DAOY and T98G cell lines with an IC50 ranging from 11µM to 20µM after 24 h of treatment. Pimozide induced apoptosis in these cell lines as evaluated by FITC/Annexin assay and further validated by the cleavage of caspase 3 as well as PARP by western blot analysis. Pimozide treatment resulted in the concentration dependent decrease in the expression of GLI1, OCT4, SOX2 and NANOG in U87MG, U251MG, DAOY and T98G brain cancer cell lines, depicting a reduction in the stem cell like property of these cell lines. In addition, pimozide treatment inhibited the expression of proto-oncogene c-Myc. Inhibition of Gli1 and c-Myc demonstrates that pimozide might also inhibit the growth of cancer cells by targeting SHH signaling. Our results further demonstrated that oral administration of pimozide (25mg/kg) inhibits the brain tumors in intracranial tumor model with no significant difference in average weight of critical organs. It is important to note that pimozide is an FDA approved drug with no considerable toxicity. Overall, this study depicts that pimizode is a potential candidate to target brain tumors.

Citation Format: Itishree Kaushik, Alok Ranjan, Blake Schwettmann, Sanjay Srivastava. Pimozide suppresses the growth of brain tumor by targeting oncogenic pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5452.
Abstract 5462: Functional characterization of potential gain-of-function p53 mutants in esophageal squamous cell carcinoma
Authors: Yu Valen Z. , Lung Maria L.
Published: August 2018

One of the most frequently mutated genes reported in human cancers is TP53. Esophageal squamous cell carcinoma (ESCC) has an extremely high frequency of p53 mutations of 60-90% in ESCC patients. Evidence of gain-of-function (GOF) p53 mutations that exert wild-type (WT) function-independent oncogenic effects are emerging. We aim to determine the functional role of several candidate GOF p53 mutations in ESCC and explore their clinical utility for prognostication and as potential treatment targets. In our recent study of Hong Kong ESCC specimens, we detected TP53 alterations in over 90% of ESCC tumors, ~36% of which are candidate GOF mutations verified in other cancers. By analyzing published ESCC sequencing data, we showed that patients from the high-risk ESCC region show frequent candidate GOF p53 mutants. We applied the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to functionally knockout (fKO) mutant p53 protein expression in various ESCC cell lines with different endogenous p53 mutants and subject them to in vivo and in vitro functional assays. Interestingly, only KYSE150 cells with an endogenous p53R248Q mutation show reduced tumor growth in nude mice after p53-fKO, suggesting that there is a specific mutant p53 that confers oncogenic features in ESCC. Consistently, only KYSE150 p53-fKO cells show reduced colony formation in three-dimension in vitro culture. Tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) is significantly reduced in KYSE150 p53-fKO cells, suggesting that regulation of the STAT3 pathway contributed to the oncogenic feature of the GOF p53 mutant. p53R248Q exhibits concentrated subcellular localization to the membrane fraction (including ER, mitochondria, etc.) and shows increased resistance to HSP90/HDAC inhibition-induced protein degradation, as compared to other p53 mutants. Together, these results suggest a GOF role of certain mutant p53 proteins with specific characteristics. Targeting such mutants and/or related oncogenic pathways may be of therapeutic benefit. Acknowledgement: We acknowledge the Research Grants Council of Hong Kong Special Administrative Region for General Research Fund support (17109515 to M.L.L).

Citation Format: Valen Z. Yu, Maria L. Lung. Functional characterization of potential gain-of-function p53 mutants in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5462.
Abstract 5466: CXCL14 suppresses the aggressive phenotype in triple-negative breast cancer (TNBC) through CXCR4
Authors: Gibbs Carla R. , Ignacio Rosa Mistica C. , Richmond Ann , Son Deok-Soo
Published: August 2018

The chemokine network is involved in cancer metastasis by modulating cell migration and angiogenesis. CXCL12-CXCR4 is a common signaling axis that is associated with cancer metastasis. Metastatic breast cancers often overexpress the CXCR4 receptor, whereas lung tissues express high levels of CXCL12; consequently, this is a proposed mechanism for breast cancer metastasis to the lung. CXCL14, an anti-inflammatory chemokine, has been postulated to be an alternative ligand for CXCR4, though a formal assignment of CXCR4 as the receptor for CXCL14 has not been made. Interestingly, CXCL14 competes with CXCL12, a main ligand for CXCR4, to modulate CXCL12-CXCR4 downstream activation of ERK. CXCL14 expression is downregulated in oral, colon, prostate, lung, pancreatic, cervical, kidney cancers, and in TNBC when compared to other subtypes of breast cancer. Overexpression of CXCL14 in cancer cell lines in vivo and in vitro leads to decrease in tumor metastasis. CXCL14 overexpression in the MDA-MB-231 breast cancer cell line leads to a decrease in cell growth, invasion and migration. To further explore a role for CXCL14 in breast cancer aggressiveness, we examined the effects of CXCL14 on the cell migration and metastasis of additional breast cancer cell lines. Here we performed cell migration and MTT cytotoxicity assays using MDA-MB-231 and BT-549 TNBC cell lines and the MCF-7 estrogen receptor+ cell line. Our results demonstrate TNBC cells are significantly more sensitive to CXCL14 compared to non-TNBC MCF7 cells (in Boyden chamber migration assay). Also, in our experiments CXCL14 did not alter proliferation in either TNBC cells or non-TNBC cells. However, when we generated CXCR4-overexpressing breast cancer cell lines using parental BT-549, MDA-MB-231, and MCF-7 cells, we observed the changes in the phosphorylation of ERK1/2 (MAPK targets) and pAKT (PI3K downstream effector protein). Based on these data, we propose CXCL14 as a negative modifier of the CXCL12-CXCR4 axis in TNBC. Based on these data, we propose a negative role for CXCL14 in the modulation of TNBC aggressiveness.

Citation Format: Carla R. Gibbs, Rosa Mistica C. Ignacio, Ann Richmond, Deok-Soo Son. CXCL14 suppresses the aggressive phenotype in triple-negative breast cancer (TNBC) through CXCR4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5466.
Abstract 549: The CUG-translated WT1, not AUG-WT1, is an oncogene
Authors: Lee Kun Yeong , Kim Hong-Gyum , Ryu Joohyun , Lim Do Young , Chen Hanyong , Bode Ann M. , Dong Zigang
Published: August 2018

The Wilms' tumor 1 (WT1) gene is believed to act as a canonical tumor suppressor. However, it has also been reported to function as an oncogene. Germline WT1 deletion is associated with Wilms' tumor, and exogenous WT1 cDNA introduction into cells induces the transcriptional suppression of its oncogenic target genes. In contrast, high WT1 expression is associated with poor prognosis in patients with various cancers. Why WT1 acts as a tumor suppressor under certain conditions but as an oncogene under other conditions is unknown. Here, we report that CUG initiation site for WT1 protein synthesis (CUG)-translated WT1 (cugWT1), an N-terminally extended form of canonical AUG initiation site for WT1 protein synthesis (AUG)-translated WT1 (augWT1), was overexpressed in most cancer cell lines and cancer tissues and functioned as an oncogene, whereas the classical augWT1 acted as a tumor suppressor as reported previously and inhibited the function of cugWT1. Translation of cugWT1 is initiated from a CUG codon upstream and in-frame with the coding region of augWT1. cugWT1 induced cell transformation and increased the gene expression of c-myc, bcl-2 and egfr, whereas overexpression of augWT1 repressed colony formation of cancer cells and inhibited the expression of the same target genes by recruiting histone deacetylase 1 (HDAC1). In addition, we found that protein kinase B (AKT)-phosphorylated cugWT1 on Ser62 and protected cugWT1 from proteasomal degradation induced by the F-box/WD repeat-containing protein 8 (FBXW8). These results provide an important breakthrough in the field of cancer biology and contribute significantly to the resolution of the chameleon function of WT1.

Citation Format: Kun Yeong Lee, Hong-Gyum Kim, Joohyun Ryu, Do Young Lim, Hanyong Chen, Ann M. Bode, Zigang Dong. The CUG-translated WT1, not AUG-WT1, is an oncogene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 549.
Abstract 5490: Targeting mitochondrial function in lung tumor growth and progression
Authors: Kalainayakan Sarada Preeta , Ghosh Poorva , Dey Sancharika , Liu Li , Zhang Li
Published: August 2018

Contrary to Warburg's hypothesis, mitochondrial oxidative phosphorylation contributes significantly to fuel cancer cells. Besides oxidative phosphorylation, mitochondria are also involved in heme synthesis. Heme serves as a prosthetic group for several proteins that constitute the complexes of mitochondrial electron transport chain. Therefore, heme plays a pivotal role in mitochondrial oxidative phosphorylation. Recently, our lab demonstrated that non-small cell lung cancer (NSCLC) cells exhibit intensified mitochondrial respiration. Targeting increased mitochondrial respiration with therapeutic agents effectively hampers proliferation of NSCLC cells in vitro. Previous results from our lab also demonstrated that NSCLC cell lines exhibited elevated heme synthesis, heme uptake, and expression of hemoproteins. These results suggest that increased mitochondrial respiration is facilitated by excess intracellular heme that is incorporated into hemoproteins. Our study aims to assess whether targeting mitochondrial function and heme synthesis affects NSCLC tumor growth and progression in vivo. To this end, luciferase-expressing NSCLC cells were implanted in NOD/SCID mice to generate lung orthotopic xenografts. The mice bearing orthotopically implanted NSCLC tumors were treated with various agents that target mitochondrial function alone (MTA1 or MTA2) and in combination with heme synthesis inhibitor. Tumor growth was monitored by noninvasive bioluminescence imaging (BLI). Our BLI data demonstrated that there was a significant reduction in radiance in mice that received the mitochondria-targeting agent alone (MTA1) and a combination of mitochondrial-targeting agents (MTA1 and MTA2). BLI data also demonstrated that there was a considerable reduction in radiance in mice that received combination of MTA and heme synthesis inhibitor. Treatments with combination of MTAs and heme inhibitor are under way. Our Immunohistochemistry data indicate that there was a reduction in expression of proteins involved in heme transport as well as hemoproteins involved in mitochondrial respiration in mice treated with the mitochondria-targeting agent. Our results suggest that targeting mitochondria significantly inhibits lung tumor growth by affecting proteins involved in heme synthesis and degradation, and consequently decreasing mitochondrial respiration.

Citation Format: Sarada Preeta Kalainayakan, Poorva Ghosh, Sancharika Dey, Li Liu, Li Zhang. Targeting mitochondrial function in lung tumor growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5490.