bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5206: Role of super-enhancers in HPV+ head and neck squamous cell carcinoma
Authors: Zamuner Fernando T. , Vorontsov Ilya , Flam Emily , Mukhina Vera , Danilova Ludmila , Ou Tingting , Stavrovskaya Elena , Guo Theresa , Windsor Eric , Kelley Dylan Z. , Parfenov Michael , Considine Michael , Fertig Elana J. , Favorov Alexander , Gaykalova Daria A.
Published: August 2019

Head and neck squamous cell carcinomas (HNSCC) are the sixth leading cause of cancer worldwide with different incidences, mortalities, and prognosis for different subsites. Infection by Human Papilloma Viral (HPV) can cause the development of HPV+ HNSCC, the most rapidly growing population of cancer patients. The molecular biology of HPV+ HNSCC is related to abnormal transcriptional regulation. Super-enhancers (SEs) were recently identified as critical regulators of gene expression during cell differentiation and disease development. SEs are long clusters of enhancers that recruit master transcription factors (TFs) and coactivators to regulate the expression of target genes. We have recently developed a new whole-genome analytical pipeline to optimize ChIP-Seq procedures on primary surgical samples, and this protocol helped us to define the whole-genome distribution of H3K27ac, the hallmark of SEs, in HPV+ HNSCC samples. To detect SEs, we used a novel LILY algorithm that corrects the ChIP-Seq signal for copy number variation and CpG density. The analysis revealed that HPV+ HNSCC-specific SEs regulate genes critical for head and neck cancer development, such as EGFR, TP63, JAG1, IRF6, SMAD3, MAP4K2, SNAI1, TNFAIP3, ANO2, and TNFRSF1A. These genes are located in the vicinity of HNSCC-specific SEs, and the expression of those genes was altered by JQ1, the inhibitor of BRD4, the main component of SE machinery, supporting the role of SE in their regulation. The following Cistrome analysis allowed us to elucidate the TF composition of the SE machinery. Thus, we have found out that P63, P53, SOX2, FOSL1, SMAD3, and SNAI2 are TFs that are the most overrepresented in the HPV+ HNSCC-specific SEs. These comprehensive analyses have revealed novel insight into the HPV+ HNSCC biology and paved the basis for the implications for epigenetic therapeutics for this tumor types, and potentially other virus-related malignancies.

Citation Format: Fernando T. Zamuner, Ilya Vorontsov, Emily Flam, Vera Mukhina, Ludmila Danilova, Tingting Ou, Elena Stavrovskaya, Theresa Guo, Eric Windsor, Dylan Z. Kelley, Michael Parfenov, Michael Considine, Elana J. Fertig, Alexander Favorov, Daria A. Gaykalova. Role of super-enhancers in HPV+ head and neck squamous cell 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 5206.
Abstract 5208: Genome-wide AR enhancer activity in prostate cancer
Authors: Ozturan Dogancan , Huang Flora , Morova Tunc , Saffarzadeh Mohammadali , Lack Nathan A.
Published: August 2019

Prostate cancer (PCa) is one of the most commonly diagnosed forms of cancer with one out of every nine North American men developing the disease in their lifetime. Despite robust early detection methods, new therapeutics, and advancements in surgical operations, PCa is still second leading cause of cancer death in men. An increasing number of studies have shown that non-coding mutations plays a critical role in the development and progression of PCa. These somatic mutations can disrupt regulatory elements such as enhancers and alter the transcriptional landscape of the cancer. Discovering and interpreting the non-coding elements is crucial to understand the nature of this disease.

At all stages of PCa, androgen receptor (AR) signaling is essential to the cancer. Recent work from our laboratory demonstrated that there is a significant increase in somatic mutations at AR binding sites (ARBS). Given the importance of AR-mediated transcription, as well as the recurrent nature of these mutations, we proposed that these somatic mutations could play an important role in PCa development. To stratify the mutations, we conducted a massively multi-parallel enhancer assay to identify those ARBS sites with enhancer activity. With STARR-seq we found that 8% of the ARBS had strong inducible AR enhancer activity (341/4139). Interestingly, the majority of these sites did not contain a canonical androgen response element. With these results we incorporated high-throughput chromosome conformation capture (HiC) to formally define the genes regulated by each of these AR enhancers. Based on these results we have selected and characterized several non-coding ARBS mutations. We believe that the importance of the regulatory role of these regions will play a tremendous role in understanding of the prostate cancer and its progression.

Citation Format: Dogancan Ozturan, Flora Huang, Tunc Morova, Mohammadali Saffarzadeh, Nathan A. Lack. Genome-wide AR enhancer activity in 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 5208.
Abstract 5212: Ablation of SMAD4 repression on Elf3 promotes the progression of lung cancer in vivo
Authors: Liu Jian , Wang Tianyuan , Wu San-pin , Li Jian-liang , Demayo Francesco J.
Published: August 2019

Lung cancer is the leading cause of cancer death among both men and women in United States. ELF3, a transcription factor, overexpresses in human and mouse lung cancer and plays an oncogenic role in lung cancer cells1,2. Understanding the regulatory mechanisms for ELF3 may help to develop related target therapies. Ablation of Smad4 and Pten in mice results in an increase of Elf3 expression and the development of adeno-squamous carcinoma1. Results from in vivo ChIP-Seq analysis demonstrate SMAD4 occupancy near the Elf3 promoter in the pulmonary epithelium1. Therefore, we hypothesize that this SMAD4 occupancy is required to repress Elf3 expression and subsequently suppresses lung cancer progression. To test this hypothesis, we deleted this SMAD4 binding region in vivo and characterized the phenotypic consequences.

CRISPR/Cas9 was used to ablate the SMAD4 ChIP-Seq binding peak on Elf3 promoter region in mice. This resulted in mice with 12 and 17 nucleotide deletions in the Elf3 promoter region. ChIP-qPCR analysis showed that SMAD4 binding was decreased while H3K27ac bindings were increased in the Elf3 promoter region. The reduction in SMAD4 binding and the epigenetic changes in the Elf3 promoter region resulted in an increased expression of ELF3 in these lungs of these mice. Histological analysis of these mouse lungs showed the presence of pulmonary hyperplasia and the development of lung tumors. In order to identify potential interacting factors with SMAD4 in the Elf3 promoter, motif analysis of the deleted SMAD4 binding peak identified GATA2, ETS1 and OXT2 as potential interacting factors.

In summary, small disruption of Elf3 promoter region bound by SMAD4 leads to the increase of ELF3 expression with the enhanced binding of H3K27ac and the decrease of SMAD4 binding and promotes lung hyperplasia and lung cancer development. SMAD4 may interact with GATA2, ETS2 and OXT2 in the regulation of Elf3expression.

Key references

1. Liu, J. et al. ErbB2 Pathway Activation upon Smad4 Loss Promotes Lung Tumor Growth and Metastasis. Cell Rep, doi:10.1016/j.celrep.2015.02.014 (2015).

2. Wang, H. et al. Overexpression of ELF3 facilitates cell growth and metastasis through PI3K/Akt and ERK signaling pathways in non-small cell lung cancer. Int J Biochem Cell Biol 94, 98-106, doi:10.1016/j.biocel.2017.12.002 (2018).

Note: This abstract was not presented at the meeting.

Citation Format: Jian Liu, Tianyuan Wang, San-pin Wu, Jian-liang Li, Francesco J. Demayo. Ablation of SMAD4 repression on Elf3 promotes the progression of lung cancer in vivo [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 5212.
Abstract 5216: Identification of Sox2 regulatory factors in small cell lung cancer
Authors: Wollenzien Hannah , Yousef Mira , Voigt Ellen , Kareta Michael S.
Published: August 2019

Sox2 is one of the core pluripotency genes that has also been implicated to be oncogenic in a multitude of cancers. We have shown that Sox2 is required for the formation of SCLC using mouse models and human cell culture. SCLC predominantly arises from in the lung from pulmonary neuroendocrine cells, which are formed from Sox2-expressing progenitors that are then silenced upon terminal differentiation. However, upon transformation Sox2 becomes highly upregulated in SCLC. One means of repressing Sox2 is the Rb tumor suppressor, which binds to the Sox2 locus and represses transcription by the recruitment of many epigenetically silencing factors. However, there are still some gaps in our knowledge of the repression of Sox2 by Rb, such as which factors are recruiting Rb to Sox2. We describe a novel technique of Cre-mediated Protein-DNA Immunoprecipitation (CrePI) to identify proteins at the Sox2 locus in an unbiased manner to further elucidate the mechanism of Rb repression of Sox2.

Citation Format: Hannah Wollenzien, Mira Yousef, Ellen Voigt, Michael S. Kareta. Identification of Sox2 regulatory factors in small cell lung 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 5216.
Abstract 522: PI3K/Akt signaling in tumor cells promotes immune evasion by limiting infiltration, recognition and killing by T cells
Authors: Sivaram Nithya , McLaughlin Patrick , Jiang Ya-Ping , Ballou Lisa , Pham Kien , Liu Chen , Velden Adrianus van der , Lin Richard
Published: August 2019

The presence of tumor-infiltrating T cells is associated with favorable patient outcomes, yet most pancreatic cancers are immunologically silent and resistant to currently available immunotherapy. In this study, a syngeneic orthotopic implantation model of pancreatic cancer was used to show that PI3K/Akt signaling in tumor cells down-regulate immunogenicity to evade T cell surveillance. Genetic silencing of PI3K catalytic isoform Pik3ca in KrasG12D/Trp53R172H-driven pancreatic tumors leads to infiltration of T cells, complete tumor regression, and 100% survival of immunocompetent host mice. By contrast, Pik3ca-null tumors implanted in T cell-deficient mice progress and kill 100% of the animals. Adoptive transfer of tumor antigen-experienced T cells eliminates Pik3ca-null tumors in immunodeficient mice. Pik3ca silencing or inhibition of its effector, Akt, increases the expression of MHC Class I and CD80 on the surface of murine and human pancreatic cancer cells. Down-regulating MHC Class I and CD80 expression, or increasing Akt activity, in Pik3ca-null tumors increases their susceptibility to T cell-mediated regression in vivo. These results indicate that PI3K/Akt signaling in tumor cells limits T cell recognition and clearance of pancreatic cancer. Strategies that selectively target the PI3K/Akt signaling pathway in tumor cells may improve immunotherapy for pancreatic cancer.

Citation Format: Nithya Sivaram, Patrick McLaughlin, Ya-Ping Jiang, Lisa Ballou, Kien Pham, Chen Liu, Adrianus van der Velden, Richard Lin. PI3K/Akt signaling in tumor cells promotes immune evasion by limiting infiltration, recognition and killing by T 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 522.
Abstract 5227: Chromatin folding disruptions in human cancers
Authors: Akdemir Kadir C.
Published: August 2019

Genomic material within the nucleus is folded into successive layers in order to package and organize the long string of linear DNA. This hierarchical level of folding is closely associated with transcriptional regulation and DNA replication. Genes within the same folding domain demonstrate similar expression and histone-modification profiles. Therefore, boundaries separating different domains have important roles in reinforcing the stability of these domain-wide features. Indeed, domain boundary disruptions in human genetic disorders or human cancers lead to misregulation of certain genes, due to de novo enhancer exposure to promoters. However, the frequency of boundary disruptions in human cancers, and whether there are recurrently affected boundaries in specific cancer types, remains unclear. Here, to understand effects and distributions of somatic structural variations (SVs) across TADs, we utilized 288,457 high-confidence somatic structural variations from 2658 tumor-normal pair high-coverage whole genome sequencing datasets across various cancer types. We comprehensively profiled structural variations effects on the domain boundaries and the regulation of genes in human cancers. Our findings demonstrate that the impact of TAD disruptions are substantially varied across tumor types and these events are reflective of overall SV burden and type. Notably, most of the TAD disruptions could result in appreciable changes in nearby gene expression in cancer cells. In addition, we demonstrated that SVs can lead to fusion of discrete TADs. Notably, complex rearrangements drastically change chromatin folding maps in the cancer genomes.

Note: This abstract was not presented at the meeting.

Citation Format: Kadir C. Akdemir. Chromatin folding disruptions in human cancers [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 5227.
Abstract 5231: Regulatory mechanism of carboxylesterase 2 expression and its role in human colorectal cancer
Authors: Ishimine Momoko , Lee Hyeon-Cheol , Nakaoka Hirofumi , Orita Hajime , Kobayashi Toshiyuki , Inoue Ituro , Sato Koichi , Yokomizo Takehiko
Published: August 2019

Irinotecan (camptothecin-11 (CPT-11)) is an anticancer drug that has been used in the treatment of a wide spectrum of cancer, and irinotecan-containing regimens such as FOLFIRI and FOLFOXIRI are standard chemotherapy regimens for advanced colorectal cancer. Irinotecan is a prodrug that is converted to its active metabolite 7-ethyl-10-hydroxycamptothesin (SN-38) by carboxylesterases CES1 and CES2. CES2 has a far higher hydrolytic efficiency against irinotecan than CES1. Since CES2 expression is higher than other CES isozymes in colon tissue, it might contribute to local (i.e., intratumoral) activation of irinotecan. Recent studies have shown that CES2 expression is regulated by p53 in human colon carcinoma cell lines. TP53 is a tumor suppressor gene and the frequency of its mutations in colorectal cancer is approximately 50%. Therefore, TP53 mutation may cause downregulation of CES2 expression and affect the sensitivity of colorectal cancer to irinotecan. In our previous study, we showed that both CES2 expression and carboxylesterase activity were significantly lower in most colorectal cancer specimens than in adjacent normal tissue. Nevertheless, there was no clear relationship between CES2 expression and TP53 gene status. We also found that p21 expression was significantly reduced in the tumors compared with adjacent normal tissue independently of their TP53 mutational status. Notably, there was a strong positive correlation between p21 and CES2 expression even in the tumors with nonfunctional p53. Recent studies have reported that colorectal tumors developed in proximal colon (right side) and distal colon (left side) exhibit different molecular characteristics and histology. Patients with left-sided colorectal tumors respond well to conventional adjuvant chemotherapies, and the prognosis of these patients is better than that of patients with right-sided colorectal tumors. To test whether location of tumor affects CES2 expression, we compared the CES2 expression levels of tumors that arose from proximal colon with those from distal colon. We also tested the potency and the selectivity of a recently reported CES2 inhibitor. Our results provide important insights into the regulatory mechanism of CES2 expression and its role in colorectal cancer.

Citation Format: Momoko Ishimine, Hyeon-Cheol Lee, Hirofumi Nakaoka, Hajime Orita, Toshiyuki Kobayashi, Ituro Inoue, Koichi Sato, Takehiko Yokomizo. Regulatory mechanism of carboxylesterase 2 expression and its role in human colorectal 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 5231.
Abstract 5237: Development of a gene expression database of renal cell carcinoma cases by NGS-combined HiCEP to identify tumor markers
Authors: Kawaguchi Makoto , Matsuo Hirotaka , Araki Ryoko , Shimizu Seiko , Takao Mikiya , Nakayama Akiyoshi , Kitamura Yosuke , Abe Masumi , Ito Keiichi , Shinomiya Nariyoshi
Published: August 2019

Objectives: High coverage expression profiling (HiCEP) is an AFLP-based comprehensive gene expression analysis technique invented in Japan. HiCEP has two unique characteristics. First, it can detect low amounts of mRNA with high sensitivity and reliability. Second, HiCEP enables highly quantitative and reproducible mRNA expression analyses. However, it requires complicated processes, including TA cloning of isolated transcripts, to obtain sequence information on detected peaks. We performed next-generation sequencing (NGS)-combined HiCEP and tried to establish a gene expression database of renal cell carcinoma (RCC) cases to identify effective tumor markers.

Materials and methods: We collected cancerous and macroscopically non-cancerous regions from 83 RCC cases. Of these, six cases with clear cell RCC were analyzed by HiCEP. Total RNA was extracted from the cancerous and non-cancerous tissues of six clear cell RCC cases, and transcribed to cDNA. The cDNA was synthesized and subjected to digestion with the restriction enzymes MspI or MseI, followed by adapter ligation. Selective PCR by 256 kinds of primer pairs was used to amplify the HiCEP fragments, and products with fluorescently-labeled primer were then analyzed by capillary electrophoresis. HiCEP fragments were sequenced using a next-generation sequencer (ion PGM, Thermo Fisher Scientific). We compared the expression levels of HiCEP peaks in cancerous tissues with those in non-cancerous tissues.

Results: We detected several HiCEP peaks in cancerous tissues that showed five times higher expression than in normal tissues. We determined the sequences of the HiCEP fragments by NGS, and developed the first ever cancerous tissue HiCEP fragment database.

Conclusion: We successfully established an RCC gene expression database by NGS-combined HiCEP. We are now performing replication analyses and further analyses of blood samples from the same RCC cases to be able to identify diagnostic and prognostic markers of RCC.

Citation Format: Makoto Kawaguchi, Hirotaka Matsuo, Ryoko Araki, Seiko Shimizu, Mikiya Takao, Akiyoshi Nakayama, Yosuke Kitamura, Masumi Abe, Keiichi Ito, Nariyoshi Shinomiya. Development of a gene expression database of renal cell carcinoma cases by NGS-combined HiCEP to identify tumor markers [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 5237.
Abstract 5239: Cisplatin disrupts MDM2-DAXX-HAUSP complex, p53 accumulation, cell cycle regulation, and apoptosis in acute leukemia cells
Authors: Kumar Sanjay , Tchounwou Paul
Published: August 2019

Cisplatin (CDDP) is an antitumor drug successfully used in the treatment of various types of human malignancies. It enhances toxicity of arsenic trioxide (ATO) in head and neck squamous cell carcinoma cell, small cell lung cancer, ovarian cancer cells, and oral squamous cell carcinoma cells (, and chronic myelogenous leukemia cells. CDDP induces cytotoxicity, DNA damage, oxidative stress, and apoptosis in acute promyelocytes leukemia (APL) cells. However, the molecular mechanisms of CDDP-induced P53 activation, cell cycle arrest, and apoptosis in APL cells remain poorly understood. We hypothesized that CDDP inhibits proliferation of APL cells through MDM2-DAXX-HAUSP complex disruption, p53 activation leading to cell cycle arrest and apoptosis. To test hypothesis, we used three APL cell lines and mice model of APL and investigated CDDP effects on cells growth, complex disruption, cell cycle progression, and apoptosis by applying western blotting, IP, gene knockdown techniques, immune/histochemistry, and confocal imaging. We found that CDDP - induced stress signal transmitted by protein kinase (ATM, ATR) and its downstream targets (CHK1 & CHK2) phosphorylation leading to down-regulation of complex molecules expression and accumulation of p53. CDDP-induced p53 expression modulated cell cycle progression and apoptosis in APL cells, and stimulated the formation of more promyelocytes with dense granules through reduction of MDM2 expression in bone marrow cells. CDDP also activated p53 in APL mice liver tissues. This novel mechanism of action of CDDP in APL cells may help in the design of new anti-leukemic drugs for treatment of APL patients.

Acknowledgements: This research was supported by National Institutes of Health NIMHD [grant # G12MD007581], through the RCMI-Center for Environmental Health at Jackson State University.

Citation Format: Sanjay Kumar, Paul Tchounwou. Cisplatin disrupts MDM2-DAXX-HAUSP complex, p53 accumulation, cell cycle regulation, and apoptosis in acute leukemia 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 5239.
Abstract 5250: Notch signaling in ER+ breast cancer in response to endocrine therapy agents
Authors: Singleton Ciera S. , Miele Lucio , Crabtree Judy S.
Published: August 2019

Breast cancer is the most frequent cancer in women in the world, and more than 70% of breast cancer cases are estrogen receptor α positive (ER+). Standard treatment for ER+ breast cancers is endocrine therapy that consists of selective estrogen receptor modulators (SERMs) such as tamoxifen in pre-menopausal patients; aromatase inhibitors (AIs); or selective estrogen receptor down-regulators (SERDs); fulvestrant, and treatment is dependent on the menopausal state of the patient. Many luminal tumors acquire resistance to therapy and recur, leading to disease progression and mortality. Resistance may result from the activation of quiescent signaling pathways that drive cancer stem cell (CSC) activity, such as the Notch signaling pathway. Previous studies demonstrate that activation of Notch via the NOTCH1 receptor recruits ERα in the absence of estrogen to facilitate transcription of estrogen responsive genes. However, NOTCH4 is often upregulated in resistant ER+ breast cancers, driving endocrine therapy resistance via breast CSC activity. We hypothesize that NOTCH4 promotes the transcription of estrogen-responsive genes in the absence of estrogen in a selective manner that is unlike NOTCH1. Understanding how NOTCH4 regulates the transcription of estrogen responsive genes upon treatment with endocrine therapy may suggest the need for combination therapy for ER+ breast cancer that includes a Notch targeted therapy. We have measured mRNA expression of classic estrogen responsive genes in ER+ breast cancer cell lines that either overexpress NOTCH1 or NOTCH4. Our data demonstrate differential expression of estrogen-responsive genes between NOTCH1 and NOTCH4 in the presence of tamoxifen. We have also measured Notch contribution to breast cancer stem cell activity through mammosphere formation assays using ER+ breast cancer cell lines in the presence of fulvestrant and Notch signaling inhibitors. The expression of classical estrogen responsive genes from mammospheres was altered in the presence of fulvestrant, Notch signaling inhibitors, or combinations of the two treatments. Further, combination treatment led to the disruption of mammosphere formation suggesting that combination therapy in the clinic may be suitable for the prevention of endocrine resistance by inhibition of BCSC activity through NOTCH4.

Citation Format: Ciera S. Singleton, Lucio Miele, Judy S. Crabtree. Notch signaling in ER+ breast cancer in response to endocrine therapy agents [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 5250.
Abstract 5253: Regulation of estrogen related receptor α by ING4 and its potential role in breast cancer pathogenesis
Authors: Shatnawi Aymen A. , Giguere Vincent
Published: August 2019

Breast cancer is one of the most frequently diagnosed tumors in the United States that affects one in eight women during their lifetime. It is second only to lung cancer as cause of cancer death in women. The orphan members of the nuclear receptor superfamily, estrogen-related receptors ERRα and γ (ERRs) isoforms are considered the master regulators of energy metabolism and their transcriptional pathways are highly associated with the cancer phenotype. Realizing the important function of co-regulators on ERRs transcriptional activity and their impact on cancer development and progression, we have identified the tumor growth family member 4 (ING4), a protein that has been implicated in several solid tumors and physically interacts with ERRα in breast cancer cellular models and modulates its transcriptional activity. Examination of the Oncomine database revealed that both ING4 and ERRα gene expression were significantly higher in invasive ductal and lobular breast carcinomas compared to normal ones, and their increased expression is inversely correlated with the overall patient survival rate in The Cancer Genome Atlas (TCGA) database. Knocking down ING4 in a breast cancer cell line using siRNA technology significantly decreased the expression of ERRα, and overexpression of ING4 upregulates the endogenous expression of ERRα. Well-defined ERRα target genes were also regulated by ING4 in the same manner as ERRα. Also, depleting ING4 expression significantly increased breast cancer cell migration in in vitro scratch assays, while both ING4 and ERRα knockdown decreased breast cancer cell proliferation. Collectively, these findings provide evidence for positive regulation of ERRα by ING4. Understanding the mechanism by which ING4/ERRα interaction regulates cellular pathways in breast cancer, underscores the impact of such regulation on biological and pharmacological actions. Thus, they can be used for the development of improved novel pharmaceuticals that target ERRα/ING4 complex or its downstream pathways.

Citation Format: Aymen A. Shatnawi, Vincent Giguere. Regulation of estrogen related receptor α by ING4 and its potential role in breast cancer pathogenesis [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 5253.
Abstract 5264: Metabolic reprogramming with β-alanine to overcome chemotherapy resistance in pancreatic cancer
Authors: Yakoub Danny , Totiger Smitha T. , Moran Alexandra , Suwal Sujit , Pimentel Julio , O'Donell Lauren , Walls Jamie , Nagathihalli Nagaraj , Merchant Nipun
Published: August 2019

Background: Increased aerobic glycolysis (Warburg effect) leading to acidic microenvironment confers chemoresistance to weakly basic Gemcitabine, the standard treatment in Pancreatic ductal adenocarcinoma (PDAC). Metabolomic perturbations in two PDAC cell lines having differential sensitivity to Gemcitabine was investigated by profiling over 40 metabolites using 1H-NMR spectroscopy. Notably, a significant decrease in the concentration of β-alanine (βA) was observed in Gem resistant compared to Gem sensitive cell line, suggesting βA may have a role in chemotherapy response. We aimed to evaluate the role of βA supplementation in improving Gem efficacy in Gem resistant cell lines

Methods: PDAC Panc-1 cell line extracts treated with Gem with and without βA supplementation were analysed via 1H-NMR spectroscopy. Aerobic glycolysis was quantified by measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using the Seahorse XF analyser platform. Global mRNA profiling was done by Next Generation Sequencing. Proliferation, migration and cell cycle analysis was measured by Xcelligence real time cell analysis system and flow cytometry, respectively. PANC-1 and BxPC-3 tumor xenografts treated with Gemcitabine with and without βA for 4 weeks and the tumor volumes and weights were measured.

Results: βA supplementation in Gem resistant cells results in: (1) decreased basal glycolytic rate, proton leak and ATP production; (2) increased pyruvate, decreased lactic acid and NAD+ concentrations with decreased LDH-A transcription; also increased carnosine levels with a more basic pH in the conditioned media; (3) enhanced sensitivity to Gem treatment, with a significant reduction in proliferation, cell migration, and increased apoptosis and relative hENt-1 mRNA levels and (5) xenograft tumor volumes and weights were significantly reduced upon βA supplementation.

Conclusion: βA supplementation reprograms cell metabolism by normalizing energy production and reducing microenvironment acidification, thus enhancing chemo-sensitivity in Gem resistant PDAC cells and xenografts. βA can be potentially used as a co-therapeutic in PDAC.

Note: This abstract was not presented at the meeting.

Citation Format: Danny Yakoub, Smitha T. Totiger, Alexandra Moran, Sujit Suwal, Julio Pimentel, Lauren O'Donell, Jamie Walls, Nagaraj Nagathihalli, Nipun Merchant. Metabolic reprogramming with β-alanine to overcome chemotherapy resistance in 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 5264.
Abstract 5267: Unbiased metabolic profiling of atypical teratoid/rhabdoid tumors predicts sensitivity to the glutamine metabolic inhibitor 6-diazo-5-oxo-L-norleucine
Authors: Wang Sabrina Z. , Poore Brad , Alt Jesse , Price Antoinette , Allen Sariah , Orr Brent , Rais Rana , Slusher Barbara , Eberhart Charles , Raabe Eric H. , Rubens Jeffrey H.
Published: August 2019

Atypical teratoid rhabdoid tumors (AT/RT) are deadly tumors of infancy in need of new, targeted therapies. Molecular analyses have revealed 3 epigenetically distinct subgroups of AT/RT. High MYC-expressing tumors are a particularly aggressive subgroup, with a 5-year median survival of 18.5%. MYC is difficult to directly target given large, flat, featureless protein-protein binding sites. We performed the first unbiased metabolic profile of AT/RT cell models by high-performance liquid chromatography-mass spectrometry. This study revealed that high MYC-expressing AT/RT have a unique metabolic profile (Partial Least Squares-Discriminant Analysis). Pathway analysis highlights a dependence on glutamine metabolism in high-MYC expressing AT/RT. High MYC-expressing cell lines grow poorly in glutamine-free media compared to low-MYC cell lines (MTS assay, glutamine-free media vs normal media). Due to this dependence on glutamine metabolism, we hypothesized that high-MYC expressing AT/RT would be especially sensitive to glutamine metabolic inhibitors. We show that the glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON) slows high-MYC expressing AT/RT cell growth, induces high rates of apoptosis, and improves survival in orthotopic mouse models of high-MYC expressing AT/RT (p =0.0027 by log-rank test). In contrast, low-MYC expressing models are relatively insensitive to DON. In uniformly labeled glutamine metabolic analyses of AT/RT cells, DON treatment led to depletion of glutathione levels by preventing the production of glutamate. DON treatment synergized with carboplatin to kill AT/RT cells in vitro and extend the life of mice bearing AT/RT orthotopic xenografts (p< 0.001 by log rank test). We aim to translate these findings into a new clinical trial to improve survival in this deadly disease.

Citation Format: Sabrina Z. Wang, Brad Poore, Jesse Alt, Antoinette Price, Sariah Allen, Brent Orr, Rana Rais, Barbara Slusher, Charles Eberhart, Eric H. Raabe, Jeffrey H. Rubens. Unbiased metabolic profiling of atypical teratoid/rhabdoid tumors predicts sensitivity to the glutamine metabolic inhibitor 6-diazo-5-oxo-L-norleucine [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 5267.
Abstract 5269: Discovery and validation of plasma acylcarnitines for the early diagnosis of hepatocellular carcinoma
Authors: Shen Eric Yi-Liang , U Abellona , Taylor-Robinson Simon , Thursz Mark , Holmes Elaine , Nicholson Jeremy
Published: August 2019

Introduction:

Hepatocellular carcinoma (HCC) is one of the commonest and the leading cause of cancer death globally. The main cause of this high mortality rate is that patients with HCC are usually diagnosed at late stages since they have only subtle clinical presentations at early stages. This late diagnosis is a result of the requirement of high-end imaging modalities, the use of invasive procedures, and the lack of reliable tumour biomarkers. Metabolomics is the study of metabolites in an organism in response to the changes in health status. The purpose of this study was to identify and validate plasma acylcarnitines as potential diagnostic biomarkers of HCC by using techniques in the field of metabolomics.

Methods:

228 EDTA-plasma samples of healthy controls, cirrhotic patients, or treatment-naïve patients with HCC, collected from Imperial College Healthcare Tissue Bank, were used as a discovery and training set. Patient factors, including basic patient characteristics, aetiologies of HCC, and Child-Pugh scores, and data relating to tumours, such as the multiplicity, the sizes in diameter, the status of macro-vascular invasion, were collected. A targeted assay was adapted to measure concentrations of carnitine and acylcarnitines with different chain length using the TQ-S tandem quadrupole mass spectrometer. Non-parametric univariate analyses were applied to evaluate the statistical difference between HCC and cirrhosis. Logistic regression was preferred to develop diagnostic models. A Gambian cohort of 165 plasma samples was then used as an external validation.

Results:

Among sixteen measured carnitine and acylcarnitines, three acylcarnitines, namely acetylcarnitine, succinylcarnitine, and hexanoylcarnitine, statistically increased in HCC patients comparing to cirrhotic patients. A linear model incorporating these three acylcarnitines and alpha-fetoprotein was then developed. The area under the ROC curve of this multivariable regression model was 0.782 (95% C.I., 0.695-0.870) in the training set. When applied to the validation cohort, the sensitivity and specificity of this model at identifying HCC patients from cirrhosis were 82.2% and 67.8%, respectively.

Conclusion:

This report provides a comprehensive profile of the changes of carnitine and acylcarnitines among HCC, cirrhotic patients and healthy controls, and determines the value of acylcarnitines as diagnostic biomarkers for HCC.

Citation Format: Eric Yi-Liang Shen, Abellona U, Simon Taylor-Robinson, Mark Thursz, Elaine Holmes, Jeremy Nicholson. Discovery and validation of plasma acylcarnitines for the early diagnosis of hepatocellular 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 5269.
Abstract 5272: Sensitive bioluminescent assays for monitoring changes in tumor and immune cell metabolism
Authors: Leippe Donna Marie , Karassina Natasha , Valley Michael P. , Vidugiris Gediminas , Cali James J. , Vidugiriene Jolanta
Published: August 2019

The tumor microenvironment is complex, containing diverse cell populations with individual nutrient requirements. Tumor and immune cells must respond to the dysregulated and changing conditions of the tumor microenvironment, as well as compete for fuels, to remain functional and viable. As metabolism is critical for proper cell function, e.g. for effective immune cell activity, understanding the metabolic needs of these cells is essential. We have developed a series of bioluminescent metabolite detection assays for monitoring cell metabolism. The assays share a common NAD(P)H bioluminescent detection technology that can be coupled with specific dehydrogenases to monitor key metabolic pathways such as: glycolysis with glucose and lactate detection; glutamine metabolism with glutamine and glutamate measurements; and lipolysis and lipogenesis with glycerol and triglyceride measurements. The assays were developed to have high sensitivity permitting detection of low levels of metabolites (femtomole detection limit), an important feature when samples contain only a few cells or small volumes. The assays can be used in a non-lytic format by analyzing metabolites in samples of collected cell culture medium. In this way changes over time can be monitored and the cells remain intact for additional measurements, such as multiplexing with viability or cytotoxicity assays. We have used these assays to study the metabolic activity of tumor cell lines and T cells. The induction of aerobic glycolysis is an early step in the activation of T cells. Here we present an example of detecting T cell activation in vitro by monitoring glycolysis and quantitating the increase in lactate secretion over time. Activation was observed only in the presence of both glucose and glutamine, confirming the requirements for both fuels and biochemical pathways for activation. In addition to understanding fuel requirements of cells, we also used the assays to follow changes of metabolic pathways in cancer cell lines treated with small molecule compounds. Treatment with such compounds could provide a means of manipulating tumor nutrient usage and metabolite secretion in the tumor microenvironment. In our experiments, metabolite levels were found to be altered by effectors of glycolysis, oxidative phosphorylation and glutamine metabolism. Multiplexing with viability and cytotoxicity assays provided information about cell health during the experiments.

Citation Format: Donna Marie Leippe, Natasha Karassina, Michael P. Valley, Gediminas Vidugiris, James J. Cali, Jolanta Vidugiriene. Sensitive bioluminescent assays for monitoring changes in tumor and immune cell metabolism [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 5272.
Abstract 5277: Glutamine antagonists synergize with L-asparaginase in MYCN-driven rhabdomyosarcoma
Authors: Maxwell Micah J. , Poore Brad A. , Hanaford Allison , Alt Jesse , Rais Rana , Slusher Barbara S. , Eberhart Charles G. , Raabe Eric H.
Published: August 2019

Rhabdomyosarcoma is the most common soft tissue sarcoma in children. Chromosomal translocations producing fusion transcription factors (PAX-FOXO1) are associated with poor prognosis. A subset of fusion-positive rhabdomyosarcomas have downstream activation of the transcription factor MYCN, a known oncogenic driver. Attempts to inhibit directly either PAX-FOXO1 or MYCN have been unsuccessful. MYCN is known to drive tumor cell reliance on glutamine metabolism. 6-diazo-5-oxo-L-norleucine (DON) is a well-characterized glutamine analog that irreversibly inhibits glutamine-utilizing enzymes. DON was well tolerated in a phase I pediatric clinical trial, but it has never been systematically tested in rhabdomyosarcoma. We show that high MYCN confers sensitivity to DON therapy in vitro. We have also developed a targeted glutamine antagonist compound, JHU083, which is similarly effective against MYCN-driven rhabdomyosarcoma cell lines. Furthermore, DON administered by intraperitoneal injection twice weekly significantly reduces flank tumor volume in a murine xenograft MYCN-driven rhabdomyosarcoma model (mean tumor volume 757 mm3 vs. 2167 mm3 in control animals, p-value 0.0000197 by t-test). In stable isotope resolved metabolomics experiments, DON primarily prevents asparagine synthesis, depleting intracellular asparagine levels by at least 50% in MYCN-driven cell lines (p-value = 0.0003). Finally, DON combined with L-asparaginase synergistically inhibits growth of MYCN-driven rhabdomyosarcoma cell lines (CI << 1 by the Chou-Talalay method, indicating strong synergy; p-value = 0.0001). We conclude that DON depletes cellular pools of asparagine, and combination therapy with DON and L-asparaginase synergistically inhibits the growth of MYCN-driven rhabdomyosarcoma. These studies provide the preclinical justification for potential clinical trials for the use DON or DON prodrugs in combination with L-asparaginase as new therapeutic options for patients with MYCN-driven rhabdomyosarcoma.

Citation Format: Micah J. Maxwell, Brad A. Poore, Allison Hanaford, Jesse Alt, Rana Rais, Barbara S. Slusher, Charles G. Eberhart, Eric H. Raabe. Glutamine antagonists synergize with L-asparaginase in MYCN-driven rhabdomyosarcoma [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 5277.
Abstract 5292: Cell-autonomous suppression of colon carcinogenesis by type-2 cGMP-dependent protein kinase
Authors: Islam Bianca N. , Hou Yali , Sharman Sarah K. , Browning Darren D.
Published: August 2019

A growing body of evidence demonstrates that cGMP signaling has anti-tumor effects in the colon, and could harbor novel targets for colon cancer prevention. The tumor suppressive components that function downstream of cGMP remain largely understudied. In the present work we have characterized the expression of cGMP-dependent protein kinases (PKG1, PKG2) in normal and cancerous tissue from humans. PKG1 was highly expressed in both normal and tumor tissue, where it localized to supportive cells in the lamina propria and stroma (respectively). In contrast, PKG2 expression was notably decreased in tumors compared to matched normal tissue, where it was identified in the epithelium by IHC. Neither PKG isoform was detected at the RNA or protein level in colon cancer cell lines. To interrogate a potential tumor-suppressor role of PKG2 in the colon epithelium we bred PKG2 knockout (KO) mice into a C57BL/6J background. These animals exhibited increased intestinal turnover as reflected by crypt hyperplasia (Ki67) and elevated luminal apoptosis (cleaved caspase 3). When subjected to AOM/DSS treatment, the PKG2 KO mice developed almost twice the number of polyps per mouse as wild type siblings (1.75-fold, p=0.0037). Mouse colon organoids were used to confirm that PKG2 was the only isoform expressed in the epithelium, where it was observed in both proliferating and differentiating epithelial compartments. Colon organoids derived from PKG2 KO mice exhibited more rapid proliferation, and a reduced ability to differentiate compared to wild type controls. Taken together our results show that PKG2 is the central target of cGMP in the colon epithelium, where it suppresses carcinogenesis by controlling proliferation in a cell autonomous manner.

Citation Format: Bianca N. Islam, Yali Hou, Sarah K. Sharman, Darren D. Browning. Cell-autonomous suppression of colon carcinogenesis by type-2 cGMP-dependent protein kinase [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 5292.
Abstract 5302: Interplay of Parkin, a 'tumor suppressor’ in metabolic deregulation of cancer
Authors: Bhat Zafar I. , Imtiyaz Khalid , Rizvi M. Moshahid
Published: August 2019

Cancer is a disease of uncontrolled proliferation which initially arises as a result of dysregulation in some gene candidates responsible for maintaining a normal cellular state. Some genes are suppressed with a concomitant increase in the expression of certain dormant ones. The former group constitutes the tumor suppressors while as the later- the oncogenes. Parkin a PARK 2 gene product is a potent tumor suppressor, has been well reported to be down regulated in many cancer types. Besides its tumor suppressor role, it regulates the redox state of the cell and recently it was reported to be an important factor in TP53 (a master tumor suppressor protein) mediated tumor suppression provides a clue about the role of Parkin in metabolic deregulation- an emerging hallmark of cancer! To this end, here we present that how Parkin is regulated under different environmental conditions and how it governs metabolic plasticity- glycolytic phenotype and hence cellular migration.

To conclude, the results show that Parkin differentially regulates metabolism by modulating certain potent marker candidates responsible for Warburg effect and associated carcinogenesis.

[MMA Rizvi is the corresponding author for this work.]

Note: This abstract was not presented at the meeting.

Citation Format: Zafar I. Bhat, Khalid Imtiyaz, M. Moshahid Rizvi. Interplay of Parkin, a 'tumor suppressor’ in metabolic deregulation of 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 5302.