bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 847: Overexpression of long noncoding RNA FAM promotes cancer invasion and drug resistance in human liver cancer
Authors: Huang Po-Shuan , Lin Kwang-Huei
Published: August 2019

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies worldwide. Most patients with HCC have an established background of chronic liver disease and cirrhosis, with major etiological and risk factors including chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV). Long non-coding (lnc)RNAs are a class of non-protein coding transcripts longer than 200 nucleotides that regulate complex cellular functions, such as cell growth, differentiation, metabolism and metastasis. Increasing evidence has shown that lncRNAs regulate gene expression by targeting the production, splicing, decay, or translation of target mRNAs. Although deregulation of lncRNAs expression have been detected in HCC or many tumor types, there are still many novel lncRNAs have not been studied and the mechanisms underlying functional impairment and specific involvement of lncRNAs during hepato-carcinogenesis remain to be established. Here, we aimed to investigate the involvement of specific dys-regulated lncRNA-FAM and its molecular mechanism. FAM is overexpressing in HCC and correlates with tumor size, vascular invasion, and pathology stage. Overexpression FAM accelerates cell proliferation, invasion in HCC cells. Further, FAM is induced in the Doxorubicin (DOX) resistance HCC cells show in the Gene Expression Omnibus (GEO) Datasets analysis. Overexpression FAM did increase DOX-resistant in two HCC cell lines. After overexpression FAM, lysosome-associated membrane protein 2 (LAMP2) expression was enhanced which also correlated with DOX-resistant. From the co-immunoprecipitation analysis, FAM directly interacted with LAMP2 to protect its ubiquitination. Thus, FAM may play an oncogenic role during hepato-carcinogenesis.

Citation Format: Po-Shuan Huang, Kwang-Huei Lin. Overexpression of long noncoding RNA FAM promotes cancer invasion and drug resistance in human liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 847.
Abstract 854: Combination effect of low dose Cytarabine and pan-RAF inhibitor LY3009120 in AML cells with RAS mutations
Authors: PARK Jihyun , Yoon Sung-Soo , Koh Youngil
Published: August 2019

The alterations of RAS oncogenes were known to be involved in various cancers. In acute myeloid leukemia (AML), there are 6% of alterations in KRAS and 13% in NRAS. The RAS/RAF/MEK/ERK cascade has roles in promoting hematopoietic cell growth, inhibiting apoptosis, and is related to chemotherapy resistance. Fortunately, the recent advance in pharmacology suggests the advent of potentially efficacious RAS inhibitors. However, current RAS inhibitor development is focused on solid tumors. We, therefore, tried to identify if potential RAS targeting molecule would be effective in hematologic malignancies. Here we focused on the combination of LY3009120, a pan-RAF inhibitor that targets RAF with widely used antimetabolite agent cytarabine on AML cell line.

We used AML cell lines, HL-60, NB-4 which have NRAS/KRAS mutation and KG-1 as RAS wild-type control cell line. In the cytotoxicity test, we first performed a single effect of cytarabine and LY3009120 in the AML cell lines using cytotoxicity assay. Based on the single treat experiment, cytarabine and LY3009120 were simultaneously treated on three AML cell lines at the indicated concentration. Above all, there was a strong synergistic effect on combination treat of cytarabine containing LY3009120 in HL-60 (56.8%, p=0.0001) and NB-4 (66%, p<0.0001) compared with KG-1 cell line (8%).

In order to examine the changes in RAF/MAPK pathways, we demonstrated western blotting using antibodies to RAS, RAF, MAPK signaling molecules. In HL-60, the phosphorylation of MEK was slightly increased by the single treat of cytarabine or LY3009120. However, the combination treatment of cytarabine and LY3009120 decreased the phosphorylation of MEK. And more, in NB-4, the phosphorylation of ERK (T202/Y204) was not affected by single treatment while the combination treatment decreased ERK phosphorylation at low concentration of LY3009120. Furthermore, we investigated the underlying mechanisms of the combined effect, PARP protein cleavage indicating cell death was examined. PARP cleavage was observed in HL-60 and NB-4 after treatment, whereas KG-1 did not show any change in PARP after treatment.

In conclusion, we show the synergistic effect between cytarabine and LY3009120 on AML cell lines which have the mutation on RAS gene. Even at the low concentration of cytarabine, combining LY3009120 significantly increases the anti-cancer effect. With our data, we suggest that novel therapeutic strategies for AML patients with RAS mutation.

Citation Format: Jihyun PARK, Sung-Soo Yoon, Youngil Koh. Combination effect of low dose Cytarabine and pan-RAF inhibitor LY3009120 in AML cells with RAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 854.
Abstract 855: Analysis of a novel variant of HER2 detected by clinical sequencing in cancer of unknown primary
Authors: Harada Yohei , Kashiwada Tomomi , Sato Akemi , Abe Tomonori , nakashima Chiho , Sueoka Eisaburo , Kimura Shinya , Sueoka-Aragane Naoko
Published: August 2019

A patient with cancer of unknown primary underwent an NGS-based multiplex gene assays (OncoPrime). As a result, a novel variant of HER2 (E401G) which is a missense alteration located in the extracellular domain of the HER2 protein was detected. This alteration has not been reported (COSMIC, Nov 2018) and its effect on protein function is unknown. Sanger sequencing using genomic DNA from peripheral blood sample showed that the variant was a somatic variant. HER2 E401G variant was confirmed by Sanger sequencing using genomic DNA extracted from the formalin-fixed paraffin-enbedded tumor tissues. In spite of somatic variant, the variant allele frequency was relatively high (55.8%). This result suggested the amplification of HER2 gene, FISH analysis confirmed the copy number gain of HER2 gene (21 copies). The prediction tools in silico (SIFT, PolyPhen-2 and PROVEAN) suggested pathogenicity of the variant, so we are now performing the functional analysis of the mutant HER2 protein. Many variants of unknown significance have been identified through clinical sequencing, establishing a database of integrated information of variant is essential to implement precision medicine of oncology. This study was approved by the Ethics Committee and the patient provided written informed consent for the use of genomic and clinical data for research purposes.

Citation Format: Yohei Harada, Tomomi Kashiwada, Akemi Sato, Tomonori Abe, Chiho nakashima, Eisaburo Sueoka, Shinya Kimura, Naoko Sueoka-Aragane. Analysis of a novel variant of HER2 detected by clinical sequencing in cancer of unknown primary [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 855.
Abstract 857: Identification and functional analysis of FGFR2 binding proteins in diffuse-type gastric carcinoma
Authors: Shirakihara Takuya , Sakai Ryuichi
Published: August 2019

Diffuse-type gastric carcinoma (DGC) is highly invasive subtype of gastric adenocarcinomas and frequently exhibit scattered peritoneal metastasis. Previous studies have shown that genes of receptor tyrosine kinases (RTKs) such as FGFR2 or Met are amplified in DGC cell lines with high frequency. Hence, these cells exhibit oncogene addiction to sustained activity of these RTKs for maintenance of malignant phenotype. In order to gain novel insight in the downstream signaling pathway of DGC-specific RTKs, phosphoproteomic analysis was performed. Phosphotyrosine-containing proteins associated with RTKs were purified through two sequential rounds of immunoprecipitation from lysates of RTK-amplified DGC cell lines. Here we will show the initial functional data of transferrin receptor (TFRC), one of the identified proteins by MS analysis. In the DCG cells with FGFR2 amplification, TFRC proteins were found to bind FGFR2 and were phosphorylated at Tyr20. Moreover, shRNA knockdown of TFRC as well as inhibitor of FGFR2 revealed effective impairment in cell proliferation. These results suggest that TFRC has essential roles in malignancy of FGFR2-dependent DGC.

Citation Format: Takuya Shirakihara, Ryuichi Sakai. Identification and functional analysis of FGFR2 binding proteins in diffuse-type gastric carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 857.
Abstract 858: Oncogenic EGFR mutations as genomic biomarkers for cetuximab and panitumumab response in colorectal adenocarcinoma
Authors: Kim Nayoung , Cho Jeonghee
Published: August 2019

Somatic mutations of Epidermal Growth Factor Receptor (EGFR) occur in ~3% of colorectal cancer patients. Here, through systematic functional screening of 21 recurrent EGFR mutations selected from public datasets, we show that 11 colon cancer-derived EGFR mutants; G63R, E114K, R165Q, R222C, S492R, P596L, K708R, E709K, G719S, G724S and L858R, are oncogenic, and able to transform cells in a ligand-independent manner. We demonstrate that cellular transformation by these mutants requires receptor dimerization. Importantly, the EGF-induced and constitutive oncogenic potential of these EGFR mutants are inhibited by cetuximab or panitumumab in vivo and in vitro.

Taken together, we propose that a subset of EGFR mutations can serve as genomic predictors for response to anti-EGFR antibodies and that metastatic CRC patients with such mutations may benefit from these drugs as part of the first-line therapy.

Citation Format: Nayoung Kim, Jeonghee Cho. Oncogenic EGFR mutations as genomic biomarkers for cetuximab and panitumumab response in colorectal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 858.
Abstract 860: Solution structure of a dGMP fill-in G-quadruplex forms in PDGFR-b promoter
Authors: Wang Kaibo , Dickerhoff Jonathan , Wu Guanhui , Lin Clement , Yang Danzhou
Published: August 2019

Platelet-derived growth factor receptor beta (PDGFR-β) is an important cell-surface-receptor tyrosine kinase implicated in PDGF signaling pathways. The PDGFR-β signaling pathway has been demonstrated as an essential component in control of the cellular growth, proliferation, survival, motility and differentiation. Overexpression of PDGFR-β is associated with tumor growth, angiogenesis, and migration, making PDGFR-β an attractive target for anticancer therapeutics. In the PDGFR-β gene promoter, a highly GC-rich proximal promoter region -165 to -139 nt upstream of the transcriptional start site is crucial for basal promoter activity, which can form G-quadruplexes (G4s). These structures can be stabilized by the G4-interactive molecules to inhibit the PDGFR-β transcriptional activity. Our previous structure study showed that the G4 structures formed in the PDGFR-β gene promoter always contain a broken G-strand. The broken-strand structure suggests a possibility of the formation of a G-vacancy-bearing G-quadruplex (GVBQ) structure, which can be complemented and stabilized by guanine and guanine derivatives. Elevated levels of guanine and guanine derivatives/metabolites are found in tumor cells, suggesting that the GVBQs may play important regulatory roles in tumor cell pathological processes, such as PDGFR-β transcriptional regulation. Using NMR, CD, EMSA and DMS footprinting experiments, herein, we contributed the first atomic resolution structure of a dGMP fill-in G4 formed in PDGFR-β gene promoter in potassium solution. Surprisingly, our results showed that solution dGMP preferentially inserts from the 5' end tetrad instead of the G-22 position of the previously determined structure, and maybe in dynamic equilibrium with our previously reported G-quadruplex. The dGMP, like other intramolecular guanines, that forms four Hoogsteen hydrogen bonds with adjacent guanines and stabilizes by electronic coordination with potassium cation as well as the π-π stacking interaction with the middle tetrad guanine. Moreover, a series of guanine derivatives were investigated to fill in this GVBQ, and found that only these derivatives that keep the ability to form Hoogsteen hydrogen bonds in G-tetrad can serve as the substrate, providing insights into designing new guanine derivatives anticancer drugs.

Citation Format: Kaibo Wang, Jonathan Dickerhoff, Guanhui Wu, Clement Lin, Danzhou Yang. Solution structure of a dGMP fill-in G-quadruplex forms in PDGFR-b promoter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 860.
Abstract 861: Precise excision of an oncogenic allele of tumor by Adenovirus-based CRISPR/Cas9 system induces tumor regression
Authors: Koo Taeyoung , Yoon A-Rum , Bae Sangsu , Kim Jin-Soo , Yun Chae-Ok
Published: August 2019

Approximately 15% of non-small cell lung cancer patients possesses several mutations in the epidermal growth factor receptor (EGFR) gene, which plays a critical role in tumor progression. To this end, we demonstrate that an adenovirus-mediated co-delivery of Cas9 and a guide RNA targeting oncogenic EGFR mutation commonly found in lung cancer can selectively remove oncogenic allele while being inactive against wild-type EGFR allele. An EGFR mutant harboring a single-nucleotide missense mutation (CTG to CGG) was targeted as this mutation leads to generation of protospacer-adjacent motif sequence recognized by the Cas9 protein of S. pyogenes. The CRISPR/Cas9-mediated excision of mutant EGFR allele with high indel rate led to potent lung cancer cell killing. Importantly, intratumoral administration of Ad-based CRISPR/Cas9 system led to potent tumor growth inhibition and complete tumor regressions in number of mice harboring EGFR mutant xenograft tumors. Collectively, our findings show that precise and controlled excision of oncogenic mutation by Ad-mediated expression of CRISPR/Cas9 system offers a powerful genomic surgical strategy to excise oncogenic mutations to treat cancers.

Note: This abstract was not presented at the meeting.

Citation Format: Taeyoung Koo, A-Rum Yoon, Sangsu Bae, Jin-Soo Kim, Chae-Ok Yun. Precise excision of an oncogenic allele of tumor by Adenovirus-based CRISPR/Cas9 system induces tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 861.
Abstract 869: Molecular characterization of a pediatric high-grade glioma (pHGG) mouse model harboring the H3.3 G34R mutation
Authors: Whalen Matthew J. , Garcia-Fabiani Maria B. , Núñez Felipe J. , Lowenstein Pedro R. , Castro Maria G.
Published: August 2019

Pediatric high-grade glioma (pHGG) accounts for 8-12% of central nervous system tumors and has a 90% mortality rate. Consequently, novel therapies are needed to advance treatment options and improve survival rates. A means to do this is through designing a mouse model that accurately mirrors pHGG in humans to further study this type of pHGG and design treatment options. We focused on the analysis of pHGG harboring mutations on histone H3.3 at glycine 34 (H3.3 G34R) in combination with ATRX and TP53 mutations. We generated a mouse model for pHGG using the sleeping-beauty transposase system and the delivery of plasmids into the sub-ventricular zone of neonatal mice. This results in the transposon-mediated integration of the genetic lesions into the brain cells, thereby allowing the generation of tumors with the desired molecular alterations. Two groups of mice have been developed; the control group (WT H3.3 group) bears tumors generated with the NRASV12 overexpression and two shRNA that target the Tp53 and Atrx genes. The H3.3 G34R group of mice harbors tumors generated with NRASV12 expression, sh-TP53, sh-ATRX, and H3.3 G34R expression. RNA-sequencing was carried out to analyze the transcriptome signature associated with the H3.3 G34R tumor phenotype. Several genes related to the activation of the immune response were differentially upregulated in the H3.3 G34R tumors, such as Ciita, CD74, and Irfl, amongst others. Some of these deregulated genes were validated by immunohistochemistry. Understanding the tumor transcriptome and characterizing the tumor’s immune microenvironment are crucial for the development of gene and immune therapies designed to target H3.3 G34R pHGG.

Citation Format: Matthew J. Whalen, Maria B. Garcia-Fabiani, Felipe J. Núñez, Pedro R. Lowenstein, Maria G. Castro. Molecular characterization of a pediatric high-grade glioma (pHGG) mouse model harboring the H3.3 G34R mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 869.
Abstract 872: Genetic models reveal that the novel VGLL2-NCOA2 fusion oncogene leverages embryonic programs for sarcomagenesis
Authors: Kendall Genevieve C. , Watson Sarah , Xu Lin , LaVigne Collette , Murchison Whitney , Rakheja Dinesh , Tirode Franck , Delattre Olivier , Amatruda James
Published: August 2019

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer characterized by a misregulation of skeletal muscle developmental pathways. To date, identified oncogenic drivers predominantly include RAS mutations or chromosomal translocations and gene fusions between PAX3 or PAX7 and FOXO1. RNAseq analysis of sarcomas with non-canonical gene fusions has identified new potential genetic drivers of tumorigenesis that have not been rigorously functionally validated for their transformation capacity and biological activity. One new fusion is a chromosomal translocation and inversion between chromosomes 6 and 8, which acts to juxtapose two transcriptional co-activators, VGLL2 and NCOA2. This VGLL2-NCOA2 fusion was identified in congenital rhabdomyosarcoma clinical cohorts by us and others, and characterizes aggressive RMS that express MYOD and MYOG histological markers. However, evidence of VGLL2-NCOA2 transformation capacity has not been verified, hindering insights into its functional contributions to tumorigenesis. Here, we interrogate the function ofVGLL2-NCOA2 using complementary genomic patient data and zebrafish model systems. We utilized the Tol2 transposon system to express mosaic human VGLL2-NCOA2 during early development, and found that VGLL2-NCOA2 is sufficient for tumorigenesis, and results in aggressive tumors with high penetrance by 75 days of age in zebrafish. Further, the histology of zebrafish tumors resembles the human disease, and tumors express markers indicative of RMS such as myog and desma. A cross-species RNAseq of patient and zebrafish VGLL2-NCOA2 RMS tumors highlights a significant enrichment and overlap between gene expression signatures. Finally, mapping the gene expression signatures of VGLL2-NCOA2 zebrafish RMS tumors along the spectrum of zebrafish embryogenesis indicates a clustering with developmental stages corresponding to early somitogenesis, highlighting their arrested developmental nature. Hence, we have generated the first animal model of human VGLL2-NCOA2 tumorigenesis, and have applied this model to understand the biology and identify potential therapeutic targets for this newly identified disease.

Citation Format: Genevieve C. Kendall, Sarah Watson, Lin Xu, Collette LaVigne, Whitney Murchison, Dinesh Rakheja, Franck Tirode, Olivier Delattre, James Amatruda. Genetic models reveal that the novel VGLL2-NCOA2 fusion oncogene leverages embryonic programs for sarcomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 872.
Abstract 875: Targeting Nrf2 driven glutathione de novo synthesis as a novel strategy to suppresses IDH1-mutated glioma
Authors: Liu Yang , Yu Di , Cai Sabrina , Yang Chunzhang
Published: August 2019

Background: Isocitrate dehydrogenase (IDH) mutation frequently occurs in low grade glioma and secondary glioblastoma, characterizing by the abnormally catalyzation of alpha-ketoglutarate (α-KG) to D-2-hydroxyglutarate (D-2-HG), which induces CpG island methylator phenotype, as well as metabolic reprogramming and high reactive oxygen species (ROS) generation. Our previous data showed that nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a crucial role in maintaining redox homeostasis of IDH1-mutated glioma cells, while the detail mechanism remains elusive. Our recent data showed that glutathione de novo synthesis is governed by Nrf2, which makes great contribution to ROS scavenging in IDH1-mutated tumors and could serve as a potential therapeutic target.

Methods: In this study, we explored the important role of glutathione de novo synthetic pathway in maintaining ROS homeostasis by small RNA interference of key enzymes GCLC, GCLM and SLC7A11. We also investigated the correlation between Nrf2 and glutathione de novo synthetic pathway by Chromatin Immunoprecipitation. We further investigated the therapeutic value of Nrf2 inhibitor, Brusatol, in IDH1-mutated cancer both in vitro and in vivo.

Results: Our data showed that IDH1 mutation cells exhibit high level steady-state of ROS generation and detoxification. Prolonged Nrf2 activity enhanced transcriptional activity of key enzymes in glutathione de novo synthesis, such as GCLC, GCLM and SLC7A11. Targeting Nrf2 using Brusatol suppressed glutathione synthesis in IDH1-mutated cells, resulted in cytotoxic effect and suppressed xenograft growth in vivo.

Conclusions: IDH1-mutated glioma highly depends on Nrf2 activity to maintain ROS homeostasis. Enhanced glutathione de novo synthesis is an important mechanism that governed by Nrf2 and plays critical role in ROS scavenging. Targeting ROS scavenging might be a novel approach to treat gliomas with IDH1 mutation.

Citation Format: Yang Liu, Di Yu, Sabrina Cai, Chunzhang Yang. Targeting Nrf2 driven glutathione de novo synthesis as a novel strategy to suppresses IDH1-mutated glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 875.
Abstract 88: Stable luciferase expressing cell lines for in vivo xenograft and syngeneic tumor model bioluminescence imaging
Authors: Foulke John , Chen Luping , Miagkov Alexei , Turner-Gillies Elizabeth , Volpe Lysa-Anne , Tian Fang
Published: August 2019

Human and mouse cancer cell lines are used in xenograft and syngeneic models, respectively, for the in vivo study of tumor formation and development, metastases, measuring tumor burden in whole animals and monitoring response to therapeutic treatment. Whole animal in vivo imaging has been widely applied by researchers due to the ease of operation in visualizing in vivo biological events, eliminating the requirement for animal subject sacrifice, allowing for continual monitoring/imaging of a single individual animal and reducing the amount of inter-animal variation. Luciferase reporters provide a relatively simple, robust, and highly sensitive means to measure biological processes and to assess drug efficacy in animal models through in vivo bioluminescence imaging. Here we report on the generation of a large panel of cell lines which express high levels of luciferase and -have broad applications for in vitro and in vivo studies. The panel includes human and mouse cell lines, which represent various common cancer and tissue types, such as breast, colon, lung, pancreas, prostate, and skin. After introduction of a Lenti-LUC2 luciferase reporter into the parental cell lines, single cell cloning was performed to isolate stable clones with high luciferase expression. We quantified both the relative and absolute in vitro bioluminescence signals within the cells and analyzed the signal-to-noise ratio. In addition, the cell lines were characterized and authenticated using cell morphology, growth kinetics, and STR analysis. In addition, luciferase labeled human cell lines were administered into immune-deficient mice either by subcutaneous or orthotropic injection to establish xenograft models. Luciferase labeled mouse cell lines were tested in the associated breed of mice to evaluate tumor growth in the syngeneic mouse model. The bioluminescence live imaging was performed using the Xenogen IVIS imaging system and the in vivo bioluminescent radiance was analyzed. Although growth can be evaluated by both traditional caliper measurement and bioluminescence in subcutaneous mouse models, only live bioluminescent imaging enables continuous monitoring tumor metastasis or analysis of tumor growth of the intracranial brain tumor models. In summary, luciferase expressing cell lines are valuable tools for elucidating mechanisms involved in tumorigenesis, studying tumors in vivo, and screening anti-cancer compounds for drug discovery and development.

Citation Format: John Foulke, Luping Chen, Alexei Miagkov, Elizabeth Turner-Gillies, Lysa-Anne Volpe, Fang Tian. Stable luciferase expressing cell lines for in vivo xenograft and syngeneic tumor model bioluminescence imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 88.
Abstract 881: A role for microsomal glutathione transferase 1 in melanin biosynthesis in melanoma
Authors: Zhang Jie , Ye Zhi-wei , Townsend Danyelle M. , Morgenstern Ralf , Tew Kenneth D.
Published: August 2019

Higher organisms have multiple defenses against toxic chemical insult, ultraviolet light (UVR) and oxidative stress. These include thioredoxin and glutathione dependent enzymes, some of which are selenium (Se) dependent, including thioredoxin reductases, Se-dependent glutathione peroxidases, Se-independent glutathione transferases and peroxiredoxins. Microsomal glutathione transferase 1 (MGST1) shares an important and unique characteristic with Se-dependent glutathione peroxidase GPX4, namely the ability to reduce lipid hydroperoxides directly in membranes. In contrast to most other glutathione transferases and peroxidases, we previously found that deletion of MGST1 in mice was embryonic lethal, signifying some essential functional importance. We found that MGST1 was crucial for embryonic development through regulating early hematopoiesis in both zebrafish and mouse derived hematopoietic stem cells. Interestingly, after partial knock-down MGST1, the number of melanocytes located at the midline of zebrafish embryos was significantly reduced. Melanocytes are specialized cells that produce melanin, contributing to skin color, but also providing an antioxidant defense system that includes UVR - causative in the initiation of melanoma. Both mRNA and protein levels of MGST1 were markedly decreased in several amelanotic melanoma cell lines and melanogenesis was significantly decreased in B16 melanoma cells when MGST1 expression was knocked down by 60%. This was accompanied by increased UVB-induced cell death in these B16 cells. In contrast, in normal human melanocytes, recombinant adenovirus induced over-expression of MGST1 induced melanogenesis, concomitant with an increase of tyrosinase protein levels. UVB light was used to show that stimulus of melanin biosynthesis occurred in different melanoma cells in quantitative agreement with their levels of MGST1. We conclude that within the melanin synthetic pathway, MGST1 is critical in regulating conversion of dopaquinone to glutathionyl-DOPA, a precursor of pheomelanin. In the absence of MGST1, dopaquinone can cause toxicities and result in reduced or absent levels of melanin, leading to a deficiency in protection against UVR-induced DNA damage. Thus, absence of MGST1 produces embryonic lethality and influences the susceptibility of skin melanocytes to melanoma.

Citation Format: Jie Zhang, Zhi-wei Ye, Danyelle M. Townsend, Ralf Morgenstern, Kenneth D. Tew. A role for microsomal glutathione transferase 1 in melanin biosynthesis in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 881.
Abstract 883: PRDX4 as a novel target for pancreatic cancer
Authors: Jain Pallavi , Sayad Azin , Mollen Erik , Xie Michael , Brown Kevin , Moffat Jason , Hedley David , Boutros Paul , Wouters Bradley , Koritzinsky Marianne
Published: August 2019

Pancreatic cancer (PCa) is a fatal malignancy showing a 5% 5-year survival rate, creating an urgent need for new therapeutic targets. Many cancers have high production of reactive oxygen species (ROS), leaving them potentially vulnerable to targeting proteins involved in ROS metabolism. In line with this, we identified Peroxiredoxin 4 (PRDX4) as an essential gene in 26% of pancreatic cell lines by mining functional genomics datasets. PRDX4 is localized in the endoplasmic reticulum (ER) where it metabolizes H2O2. We validated in nine established PCa cell lines and patient derived primary cell lines that depletion of PRDX4 using siRNA or shRNA leads to inhibition of proliferation and cell death in a subset of pancreatic cancer cells. This was accompanied by increased levels of reactive oxygen species (ROS) as measured by flow cytometry. Dual-Depletion of NADPH oxidase 4 (NOX4) and PRDX4 could rescue the cell death phenotype thereby indicating that increase in ROS was a consequence of NOX4 activity. PRDX4 depletion led to focal accumulation of phosphorylated H2AX (γH2AX) in the cellular nuclei, consistent with DNA damage. Phosphorylation of H2AX was exclusively dependent upon activation of the upstream kinase DNA-PKcs. Cells depleted for PRDX4 were also more sensitive to ionizing radiation. Depleting PRDX4 with inducible shRNA in established xenografts inhibited tumor growth. Finally, using transcriptome data accompanying the functional genomics datasets, we identified a 2-gene expression signature that predicts sensitivity to PRDX4 targeting.

This study indicates that depletion of PRDX4 causes increased ROS resulting in DNA damage leading to DNA-PKcs activation, inhibition of proliferation and cell death in a subset of pancreatic cancers. PRDX4 is a potential novel therapeutic target that may act synergistically with radiation therapy.

Citation Format: Pallavi Jain, Azin Sayad, Erik Mollen, Michael Xie, Kevin Brown, Jason Moffat, David Hedley, Paul Boutros, Bradley Wouters, Marianne Koritzinsky. PRDX4 as a novel target for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 883.
Abstract 885: Induction of PRDX2 by H. pylori reduces ROS and promotes cancer cell survival and resistance to cisplatin
Authors: Wang Sen , Chen Zheng , Lu Heng , Zhu Shoumin , Peng Dunfa , Soutto Mohammed , Gomma Ahmed , Bhat Nadeem , Naz Huma , Xu Zekuan , El-Rifai Wael
Published: August 2019

Background & Aims: The antioxidant enzyme peroxiredoxin 2 (PRDX2) plays a critical role in regulating reactive oxygen species (ROS) levels in several diseases. Helicobacter pylori (H. pylori) infection is a well-known risk factor of gastric cancer. The role of PRDX2 in gastric tumorigenesis remains largely unknown. We investigated the molecular function and regulation of PRDX2 in response to infection with H. pylori and cisplatin treatment in gastric cancer cells.

Methods:Western blots (WB) and quantitative real-time PCR (qPCR) analysis were performed on AGS, SNU-1 and MKN28 gastric cancer cell lines to detect PRDX2 expression levels with or without H. pyloriinfection (7.13 or J166 strain). We evaluated the levels of ROS by H2DCFDA staining. WB analysis was used to determine oxidative DNA damage and double stranded DNA breaks by using antibodies against 8-Oxo-guanine and p-H2AX. WB and NF-κb luciferase reporter assay were performed to investigate the link between PRDX2 and NF-κb signaling. ATP-GLO cell titer analysis was utilized to determine cell viability.

Results:Our data indicated that PRDX2 mRNA and protein expression levels were induced byH. pyloriin AGS and SNU-1 cells. PRDX2 knockdown significantly increased ROS levels and 8-Oxoguanine staining followingH. pyloriinfection in both cells. These results were further confirmed by Western blot data showing that p-H2AX protein level was strongly induced in PRDX2 knockdown and H. pyloriinfected cells. Interestingly, Western blot data showed that TNF-αtreatment induced PRDX2 protein levels while Bay 11-7082 treatment decreased PRDX2 protein level in AGS and SNU-1 cells. At the same time, PRDX2 transient knockdown in both AGS and SNU-1 cells decreased p-P65 (S536) protein expression levels, nuclear localization of NF-κB-p65, and luciferase reporter activity (P<0.05). We also found that inhibition of PRDX2 significantly sensitized AGS and SNU-1 cells to cisplatin treatment.

Conclusion:Our data indicates that knockdown of PRDX2 enhances ROS and DNA damage with H. pylori infection on gastric cancer cells after H. pylori infection and sensitizes gastric cancer cells to cisplatin treatment. Our results suggest a positive feedforward loop between PRDX2 and NF-kB to reduce ROS levels.

KEY WORDS: PRDX2,H. pylori, gastric cancer, reactive oxygen species, DNA damage, NF-κb

Citation Format: Sen Wang, Zheng Chen, Heng Lu, Shoumin Zhu, Dunfa Peng, Mohammed Soutto, Ahmed Gomma, Nadeem Bhat, Huma Naz, Zekuan Xu, Wael El-Rifai. Induction of PRDX2 by H. pylori reduces ROS and promotes cancer cell survival and resistance to cisplatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 885.
Abstract 887: Silver nanoparticles and ionizing radiation induce mitochondrial protein oxidation and effects on cell cycle and proliferation in lung cancer cell lines
Authors: Holmila Reetta J. , Vance Stephen A. , Singh Ravi , King S. Bruce , Furdui Cristina M.
Published: August 2019

Silver nanoparticles (AgNP) are widely applied nanomaterials for both commercial and clinical biomedical applications. AgNP have been shown to have cytotoxicity in various cancer cell lines, as well as radiosensitizing properties. The AgNPs may induce free radicals via Fenton-type reactions, but the cellular mechanisms related to reactive oxygen species (ROS) production remain largely unknown. Both ionizing radiation (IR) and AgNP exposure lead to apoptotic and necrotic cell death through mechanisms that involve DNA damage and induction of oxidative stress, possibly targeting mitochondria. Changes in the thiol redox state of mitochondrial proteins, such as protein sulfenylation, play an important role in a number of cellular processes, and mitochondrial damage and dysfunction along with increased mitochondrial ROS are implicated in a range of human diseases such as cancer. In this work we studied the molecular effects of AgNP exposure with and without IR in a panel of lung cell lines (three cancer cell lines: A549, Calu-1 and NCI-H358 and one immortalized lung epithelial cell line: BEAS-2B). Both AgNP and IR exposures increased mitochondrial protein oxidation detected by a recently developed mitochondria targeted probe for protein sulfenylation (DCP-NEt2C). This increase in DCP-NEt2C labeling was dose and time dependent and the exposures also showed a combined effect. Moreover, the exposures induced changes in cell cycle and decreased cellular proliferation. There were marked differences between different cell lines, likely reflecting differences in the cellular redox state and metabolism. In conclusion, the AgNP and IR exposures increase the mitochondrial ROS as demonstrated by increased protein oxidation and have effects on the cell cycle and cell proliferation. They also appear to have a combined effect which might have implications for clinical applications as well as environmental risk assessment.

Citation Format: Reetta J. Holmila, Stephen A. Vance, Ravi Singh, S. Bruce King, Cristina M. Furdui. Silver nanoparticles and ionizing radiation induce mitochondrial protein oxidation and effects on cell cycle and proliferation in lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 887.
Abstract 889: Biochemical and histopathological evaluation of Al2O3 nanomaterials in kidney of Wistar rats
Authors: Patlolla Anita , Tchounwou Paul
Published: August 2019

The present study was investigated to evaluate the response of kidneys in Wistar rats following long term exposure to Al2O3 nanomaterials. To achieve this objective, Al2O3 of three different sizes (30nm, 40nm and bulk) was orally administered for 28 days to 9 groups of 10 Wistar rats each at the dose of 500, 1000 and 2000 mg/kg/rat. A tenth group of 10 rats received distilled water and served as control. After 28 days of exposure the animals were sacrificed and the serum was collected and tested for the activity levels of creatinine and urea following standard methods. Induction of oxidative stress was also investigated by assessing TBARS (MDA), Protein carbonyl, reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) activities. A Histopathological evaluation was also performed to determine the extent of kidney damage. The results showed that both serum creatinine and serum urea levels increased significantly in the treated rats compared to control animals. The increase was found to be more in Al2O3-30nm followed by Al2O3-40nm and Al2O3-bulk treated rats in a dose-dependent manner. Further administration of Al2O3 significantly increased the activities of TBARS, protein carbonyl, catalase and decreased the activities of GSH and SOD in a dose dependent manner in the kidney of rats compared with control group. Histopathological evaluation showed significant morphological alterations in kidney tissue of treated rats in accordance with biochemical parameters. Taken together, the results of this study demonstrate that Al2O3 is nephrotoxic and its toxicity may be mediated through oxidative stress. Further, the results suggest that prolonged oral exposure to Al2O3 NMs has the potential to cause biochemical and histological alterations in kidney of rats at high concentration.

Note: This abstract was not presented at the meeting.

Citation Format: Anita Patlolla, Paul Tchounwou. Biochemical and histopathological evaluation of Al2O3 nanomaterials in kidney of Wistar rats [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 889.
Abstract 890: Nuclear factor kappa B (NF-κB) promotes oxidative stress adaptation and survivorship
Authors: White Elshaddai , Carter Jada R. , Hinton Cimona V.
Published: August 2019

Cellular oxidative stress is one of the several hallmarks of the aggressive phenotypes observed in prostate cancer. As levels of reactive oxygen species (ROS) increase, oxidative stressors activate mechanisms allowing cancer cells to adapt and survive. It is unclear how cancer cells adapt and survive oxidative stress; however, literature demonstrates that the transcription factor, nuclear factor-kappa B (NF-κB) contributes to survival during oxidative stress. In cancer, several factors resulting in oxidative stress, including chemotherapy, correlate with NF-κB expression and transcriptional activity, enhancing tumor survivorship and resistance to therapies. We examined adaptability and survivorship to oxidative stress in two common prostate cancer models, DU145 and PC3, which display varying degrees of tumorigenicity, respectively. We observed that serum-starvation induced ROS, stimulating an early oxidative stress environment, and cells further conferred adaptability with the addition of hydrogen peroxide (H202). Measurement of cell viability demonstrated a low death profile in stressed cells, while cell proliferation was stagnant. Quantitative measurement of apoptosis showed no significant cell death in stressed cells suggesting an adaptive mechanism to oxidative stress survival. Consistent with stress adaptation, we observed NF-κB in nuclear fractionations of cell lines which were consistent with nuclear localization observed in other diseases in literature that adapt to oxidative stress. Adaptability to oxidative stress may be a precursor to the inflammatory response in prostate tumor tissues, and/or a general survival mechanism to anti-tumorigenic agents.

Acknowledgements: These studies were supported by the NIH/NIGMS/RISE #5R25GM060414-15 and NIH/NIHMD/RCMI #5G12MD007590.

Citation Format: Elshaddai White, Jada R. Carter, Cimona V. Hinton. Nuclear factor kappa B (NF-κB) promotes oxidative stress adaptation and survivorship [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 890.
Abstract 902: The antiandrogen bicalutamide induces senescence in LNCaP cells and quiescence in Myc CaP cells
Authors: Carpenter Valerie J. , Saleh Tareq , Gewirtz David A.
Published: August 2019

The survival benefits in prostate cancer (PCa) conferred by androgen deprivation therapies (ADT) and antiandrogens are often blunted by the development of castration resistant disease (CRPC). There is increasing evidence that the primary cellular response to ADT is senescence rather than apoptosis, and this may influence progression to CRPC. However, the majority of in vitro studies employ the charcoal stripped serum model, and very few studies use antiandrogens, despite the fact that antiandrogens are currently one of the primary modes of therapy for prostate cancer. In an effort to enhance our understanding of antiandrogen effects in PCa, we evaluated the cellular response to the clinically relevant antiandrogen, bicalutamide, in the human LNCaP and murine Myc-CaP cell lines. Acute treatment (48 hours) with bicalutamide resulted in a growth arrest followed by proliferative recovery in both cell lines. However, while the growth arrest was prolonged (lasting at least 6 days) in the LnCaP cells, growth arrest was exceedingly brief (lasting approximately 24 hours) in the Myc-CaP cells. Consistent with these observations, we were able to detect increased beta galactosidase staining (a hallmark of senescence) in the LnCaP treated cells but not the Myc-CaP treated cells. Despite the fact that profound morphological changes were evident in the Myc-Cap cells, including an enlarged and elongated cell body, the highly transient nature of the growth arrest suggests that the Myc-Cap cells undergo quiescence rather than senescence. The absence of senescence induction in the Myc-CaP cells may be a consequence of the androgen receptor copy number gain. However, androgen duplication is highly uncommon in human prostate cancers prior to development of CRPC. Thus, it is likely that antiandrogens can induce senescence in treatment-naïve patients, as seen in the LNCaP cell line. The ability of bicalutamide to induce senescence, albeit perhaps conditionally, suggests that some prostate cancers treated with ADT and/or antiandrogens may be susceptible to drugs that target senescent cells (senolytics). We therefore propose that elimination of senescent cells prior to recovery could attenuate or prevent the development of CRPC.

Citation Format: Valerie J. Carpenter, Tareq Saleh, David A. Gewirtz. The antiandrogen bicalutamide induces senescence in LNCaP cells and quiescence in Myc CaP cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 902.
Abstract 914: Development of a whole-urine, next-generation sequencing-based assay for early detection of aggressive prostate cancer
Authors: Cani Andi K. , Hu Kevin , Siddiqui Javed , Han Sumin , Hovelson Daniel H. , Liu Chia-Jen , Salami Simpa S. , Palapattu Ganesh S. , Morgan Todd M. , Wei John T. , Chinnaiyan Arul M. , Tomlins Scott A.
Published: August 2019

Despite advances in biomarker development, early detection of aggressive prostate cancer (PCa) remains challenging. Existing biomarkers show modest improvement over models based on serum prostate specific antigen (PSA). We have previously developed a clinical-grade laboratory-developed test, named MiProstate Score (MiPS), for individualized risk prediction of aggressive prostate cancer. It uses transcription-mediated amplification to quantify the gene-fusion TMPRSS2:ERG (T2:ERG) (T1E4 splice isoform) and the lncRNA PCA3 from whole-urine obtained after a digital rectal exam (DRE), combined with serum PSA. To improve MiPS, we describe here the pre-clinical development and validation of a targeted next generation sequencing assay (NGS-MiPS) using post-DRE urine RNA to asses ~90 PCa transcriptomic biomarkers. These include those in MiPS as well as many isoforms of common PCa gene fusions, mRNA, and lncRNA candidate biomarkers nominated by our large-scale PCa tissue RNAseq and other sources. We have obtained a 98% informative sample rate from 2.5 mL of urine and high technical reproducibility (Pearson r=0.99). Risk scores for having PCa [or high-grade PCa (Gleason Score >6)] on biopsy, as determined by clinical MiPS vs. the clinical MiPS model using NGS data, were highly concordant, Pearson’s r=0.74 (and r=0.81). Urine from patients with benign or Gleason 6 vs. Gleason ≥ 4+3=7 cancer on biopsy (extreme design) showed expected differences in the levels of T2:ERG T1E4 (p=0.00003) and PCA3 (p=0.07), with additional T2:ERG splice isoforms and other biomarkers also being significantly different between low vs. high grade cancer. Feature selection and logistic regression trained in an extreme design cohort (n=73) yielded a 29-transcript model that outperformed MiPS and serum PSA in two validation cohorts: 1. A held-out set from the extreme design cohort n=36, AUC 0.81 vs. 0.76 and 0.63 respectively; 2. A separate active surveillance cohort n=45, AUCs 0.66 vs. 0.56 and 0.53 respectively. These results support the potential utility of our urine based targeted NGS assay to supplement serum PSA for the early detection of aggressive prostate cancer.

Citation Format: Andi K. Cani, Kevin Hu, Javed Siddiqui, Sumin Han, Daniel H. Hovelson, Chia-Jen Liu, Simpa S. Salami, Ganesh S. Palapattu, Todd M. Morgan, John T. Wei, Arul M. Chinnaiyan, Scott A. Tomlins. Development of a whole-urine, next-generation sequencing-based assay for early detection of aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 914.