bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1337: BH3-mimetic gossypol promotes autophagy to inhibit the proliferation of mutant BRAF melanoma cells with high expression of p21Cip1
Authors: Lee Michael , Jang Gun-Hee , Ahn Jun-Ho , Kim Na-Yeon
Published: May 2015

Gossypol is a BH3-mimetic small-molecule pan-inhibitor of Bcl-2. In this study, we described the potential therapeutic effects of gossypol on BRAF inhibitor-resistant melanoma cells. We found that gossypol retained its efficacy in mutant melanoma clones resistant to BRAF inhibitor through a mechanism independent of MEK-ERK inhibition. Gossypol caused G2/M arrest in BRAF mutant A375P cells with high expression of p21Cip1, regardless of their drug resistance. Interestingly, we determined that the lack of gossypol-induced mitotic arrest in BRAF-WT-harboring SK-MEL-2 cells was associated with a low level of p21Cip1 expression. In addition, gossypol preferentially induced autophagy and apoptosis in the gossypol-sensitive cells and not in the gossypol-resistant SK-MEL-2 cells, which suggests that apoptosis and autophagy can occur simultaneously and act as cooperative partners for the induction of cell death. Taken together, these results suggest that gossypol may exhibit potential for the treatment of BRAF inhibitor-resistant tumors, but a functional p21Cip1 is a prerequisite for a positive response to its clinical application.

Citation Format: Michael Lee, Gun-Hee Jang, Jun-Ho Ahn, Na-Yeon Kim. BH3-mimetic gossypol promotes autophagy to inhibit the proliferation of mutant BRAF melanoma cells with high expression of p21Cip1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1337. doi:10.1158/1538-7445.AM2014-1337
Abstract 134: HNF4α inhibits liver cancer metastasis via suppression of NF-кB activity
Authors: Ning Bei-Fang , Liu Jiao , Xu Wen-Ping , Yin Chuan , Zhang Xin , Xie Wei-Fen
Published: May 2015

BACKGROUND & AIMS: Hepatocyte nuclear factor 4α (HNF4α) is a liver enriched transcription factor and is indispensable for liver development. However, the role of HNF4α in hepatocellular carcinoma (HCC) metastasis remains largely unknown.

METHODS: 429 HCC tissues were involved in the correlation analysis of HNF4α levels with tumor features and patient outcomes. Effect of HNF4α on HCC metastasis was monitored through in vivo imaging system. Expression of HNF4α, RelA(p65) or microRNAs in human HCCs was determined by Real-time PCR, western blot or immunohistochemistry. Point mutation and ChIP assay were performed to detect the interaction of HNF4α with the promoter of target microRNA. 3’UTR reporter assay was conducted to determine the regulation of RelA by microRNAs.

RESULTS: Reduced HNF4α expression correlated well with the aggressive clinicopathological characteristics of HCC and predicted poor prognosis of patients. HNF4α levels were even lower in metastatic HCCs, and ectopic HNF4α expression suppressed the metastasis of hepatoma cells both in vitro and in vivo. HNF4α suppressed HCC metastasis through inhibiting RelA expression and NFκB activity. miR-7 and miR-124, which were transcriptionally up-regulated by HNF4α, repressed RelA expression via interaction with RelA-3’UTR. Herein, HNF4α suppresses NF-κB activity and RelA expression via a miR-7 and miR-124-dependent manner. Moreover, combination of HNF4α and NF-κB exhibited more powerful predictive efficiency of patient prognosis.

CONCLUSIONS: Considering the importance of HNF4α and NF-κB in chronic liver disease, the suppression of NF-κB by HNF4α is not only essential in connecting hepatic inflammation and tumorigenesis, but also indicates novel strategy in HCC prevention and therapy.

Citation Format: Bei-Fang Ning, Jiao Liu, Wen-Ping Xu, Chuan Yin, Xin Zhang, Wei-Fen Xie. HNF4α inhibits liver cancer metastasis via suppression of NF-кB activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 134. doi:10.1158/1538-7445.AM2014-134
Abstract 1341: Rapid decrease of ATP followed by necrosis-like cell death in bladder cancer cells after exposure to high-dose chemotherapeutics used in intravesical therapy
Authors: Yoshida Takahiro , Okuyama Hiroaki , Nakayama Masashi , Nishimura Kazuo , Nonomura Norio , Inoue Masahiro
Published: May 2015

Objectives) Non-muscle invasive bladder cancer is often treated by intravesical chemotherapy following transurethral resection. Mechanism of the effect of intravesical chemotherapy remains elusive. In this experiment, we studied cell death mechanism induced by high-dose chemotherapeutics using a novel culture method for primary cancer cells.

Materials and methods) We have developed a new culture method which enables preparation and culture of primary cancer cells as multicellular spheroids which are termed cancer tissue-originated spheroids (CTOSs) (Okuyama 2013 J Urol). First we generated a bladder cancer patient-derived xenograft. We prepared CTOS from the xenograft, and exposed to high-dose (1mg/ml) or low-dose (0.01mg/ml) of epirubicin (e-ADM) or mitomycin C (MMC) for 2 hours. We investigated absorption of chemotherapeutics into CTOSs and assessed the mode of cell death. Next, we prepared CTOSs directly from surgical specimens of bladder cancer and tested the individual responses after exposure to high-dose e-ADM and MMC.

Results) At high dose, e-ADM was promptly and homogenously delivered into cancer cells in the CTOSs. High-dose e-ADM and MMC decreased ATP levels in CTOSs within an hour accompanied with mitochondrial swelling and reduction of mitochondrial membrane potential, while plasma membrane integrity was mainteined. Some of the molecules in mitochondrial apoptotic pathway were subsequently activated, while at later time point the cells lost integrity of cell membrane without DNA laddering. In contrast, CTOSs exposed to low-dose of e-ADM or MMC exhibited typical DNA laddering. Intriguingly, the decrease of ATP levels by e-ADM and MMC was varied among CTOSs derived from 20 surgical specimens.

Conclusions) High-dose chemotherapeutics used in intravesical therapy may induce necrosis-like cell death, different from typical apoptosis caused by chemotherapeutics at the dose of systemic chemotherapy. Each bladder cancer may have a different response to high-dose chemotherapeutics used in intravesical therapy, indicating necessity of chemosensitivity test aiming for personalized medicine.

Citation Format: Takahiro Yoshida, Hiroaki Okuyama, Masashi Nakayama, Kazuo Nishimura, Norio Nonomura, Masahiro Inoue. Rapid decrease of ATP followed by necrosis-like cell death in bladder cancer cells after exposure to high-dose chemotherapeutics used in intravesical therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1341. doi:10.1158/1538-7445.AM2014-1341
Abstract 1347: The role of cell-cell contacts in the survival of extracellular matrix detached mammary epithelial cells
Authors: Rayavarapu Raju , Pagani Nicholas , Heiden Brendan , Schafer Zachary T.
Published: May 2015

Normal epithelial cells require attachment to the extracellular matrix (ECM) in order to survive. When detached from the ECM, normal cells will undergo apoptosis through a process known as anoikis. The ErbB2 receptor tyrosine kinase is well known to antagonize anoikis although the precise molecular mechanisms are not entirely known. Interestingly, we have discovered that the overexpression of ErbB2 in non-malignant mammary epithelial cells (MCF-10A) results in substantial aggregation of cells that coincides with protection from anoikis. Therefore, we were interested in understanding if these aggregates could be involved in the evasion of anoikis induction. We found that the disruption of cell-cell contacts in ErbB2-overexpressing MCF-10A cells (using methylcellulose or by antagonizing the formation of adherens junctions) induces caspase activation. Furthermore, in cells that form large aggregates, ErbB2 can physically interact with E-cadherin and EGFR in a fashion that prevents the internalization of EGFR and ultimately its lysosome mediated degradation. The disruption of aggregation causes the ErbB2/EGFR/E-Cadherin complex to fall apart and subsequently results in EGFR degradation in the lysosome. This degradation of EGFR diminishes signaling through the MAPK pathway which prevents the induction of anoikis. In summary, these data suggest that oncogenic signaling through ErbB2 promotes the formation of cellular aggregates that function to prevent EGFR from degradation and subsequently to block the induction of anoikis.

Citation Format: Raju Rayavarapu, Nicholas Pagani, Brendan Heiden, Zachary T. Schafer. The role of cell-cell contacts in the survival of extracellular matrix detached mammary epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1347. doi:10.1158/1538-7445.AM2014-1347
Abstract 1356: Frequent H3F3A K27M mutations in thalamic gliomas from young adult patients
Authors: Mukasa Akitake , Aihara Koki , Gotoh Kengo , Saito Kuniaki , Nagae Genta , Tsuji Shingo , Tatuno Kenji , Yamamoto Shogo , Takayanagi Shunsaku , Narita Yoshitaka , Shibui Soichiro , Aburatani Hiroyuki , Saito Nobuhito
Published: May 2015

Introduction. Mutations in the H3F3A gene, which encodes histone H3.3, were recently reported in cases of pediatric glioblastoma. H3F3A K27M mutations occur in gliomas that arise at midline locations, including the pons, thalamus, and spine; moreover, this particular mutation is found mainly in tumors in children and adolescents. In this study, we aimed to determine the association between H3F3A mutations and adult thalamic glioma.

Methods. Genomic H3F3A was sequenced from 20 separate thalamic gliomas. Of the 20 tumors, 18 were high-grade thalamic gliomas, and of these 18, 11 were from patients under 50 years of age (median age, 38 years; range, 17 - 46), and seven were from patients more than 50 years of age. Additionally, for 14 of the 20 gliomas, 639 genes_including cancer-related genes and chromatin-modifier genes_were sequenced, and the Infinium HumanMethylation450K BeadChip was used to examine DNA methylation across the genome.

Results. The H3F3A K27M mutation was present in 10 of (91%) of the 11 younger patients, and absent from all seven older patients. Additionally, H3F3A K27M was not detected in the two diffuse astrocytomas. By additional sequencing, recurrent mutations were identified in TP53, ATRX, NF1, and EGFR. In addition, a KDM6A mutation was found in one case of diffuse astrocytoma and a CREBBP mutation was identified in one case of glioblastoma with the H3F3A K27M mutation. Gliomas with H3F3A K27M from pediatric or young-adult patients had similar, characteristic DNA methylation profiles. In contrast, thalamic gliomas with wild-type H3F3A had DNA methylation profiles similar to those of hemispheric glioblastomas.

Conclusion. We found that high-grade thalamic gliomas from young adults, like those from children and adolescents, frequently have the H3F3A K27M mutation.

Citation Format: Akitake Mukasa, Koki Aihara, Kengo Gotoh, Kuniaki Saito, Genta Nagae, Shingo Tsuji, Kenji Tatuno, Shogo Yamamoto, Shunsaku Takayanagi, Yoshitaka Narita, Soichiro Shibui, Hiroyuki Aburatani, Nobuhito Saito. Frequent H3F3A K27M mutations in thalamic gliomas from young adult patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1356. doi:10.1158/1538-7445.AM2014-1356
Abstract 1375: Epigenetic changes associated with resistance to sunitinib in human clear cell renal cell carcinoma models
Authors: ADELAIYE REMI , Miles Kiersten Marie , Ciamporcero Eric , Conroy Dylan , Ramakrishnan Swathi , Orillion Ashley , Ku Sheng Y. , Elbanna May , shen Li , Chintala Sreenivasulu , Pili Roberto
Published: May 2015

Background: Sunitinib is first-line therapy for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite the clinical efficacy of sunitinib, in the majority of patients the disease eventually develops resistance and progresses. Epigenetic modifications of histone protein in chromatins have been shown to play a role in the regulation of gene transcription patterns in cells primarily by the catalytic activity of histone methyltransferase. EZH2 has been shown to contribute to tumor angiogenesis by inactivating anti-angiogenic factors via methylation at their promoter region. In this study, we examined epigenetic changes that may be associated with response and the resistant phenotype. Methods: Human ccRCC xenograft models RP-01 (isolated from a skin metastasis in a patient with sporadic ccRCC) and RP-02 (isolated from a skin metastasis in a patient with hereditary ccRCC) were implanted (∼1mm pieces) subcutaneously into SCID mice and were randomly assigned into two groups (sunitinib and vehicle). Mice were treated 5 days/week with sunitinib at 40mg/kg. Tumor volumes and body weights were assessed weekly by caliper measurements and weigh scale, respectively. Tumor tissues and blood were collected prior treatments and when tumors became resistant to treatment. Tissues collected were used for immunohistochemistry studies and RNA analysis. Results: Our results showed that RP-01 and RP-02 tumors, although initially responsive to treatment at 40 mg/kg, eventually became resistant to treatment. Immunohistochemistry analysis of RP-01 tumor samples indicated tumors to be hypo-vasculature when responding to sunitinib, but then became hyper-vascularized at point of resistance by CD31 staining. Analysis also revealed an associated increase in the expression of the methyltransferase EZH2 when tumors became resistant to sunitinib. However, there was no change in the expression levels of its EZH2 associated histone make H3K27me3. In addition, we noticed an increase in the levels of H3k9me2 and H3k4me2 in tumors resistant to sunitinib as compared to controls. Conclusion: Overall, our results suggest the potential role of epigenetic changes that that may be associated with sunitinib-induced resistance in ccRCC

Citation Format: REMI ADELAIYE, Kiersten Marie Miles, Eric Ciamporcero, Dylan Conroy, Swathi Ramakrishnan, Ashley Orillion, Sheng Yu Ku, May Elbanna, Li shen, Sreenivasulu Chintala, Roberto Pili. Epigenetic changes associated with resistance to sunitinib in human clear cell renal cell carcinoma models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1375. doi:10.1158/1538-7445.AM2014-1375
Abstract 1380: Genome-wide fingerprinting of regulatory chromatin to evaluate the tissue specific origins of high-grade serous ovarian cancer
Authors: Shen Howard C. , Coetzee Simon , Hazelett Dennis J. , Coetzee Gerhard A. , Noushmehr Houtan , Gayther Simon A.
Published: May 2015

The normal precursor tissue origins of high grade serous epithelial ovarian cancers (HSOC) are likely to be the fallopian tube epithelium (FTE) and/or the ovarian surface epithelium (OSE). To address this hypothesis, we used FAIRE-seq and ChIP-seq to evaluate the genome wide regulatory profiles of both tissue types and compared these profiles to well characterized HSOC cell lines (CaOV3 and UWB1.289). This analysis provided, for the first time, global high-resolution annotation of open chromatin and histone H3K27 acetylation and H3K4 monomethylation marks for these tissues types, which are consistent with sites of transcriptional regulation. Using these data, we identified over 10,000 non-promoter associated FAIRE/ChIP signal sites that represent candidate functional enhancers. The regulatory features provided a cell type-specific fingerprint for each cell type. While there were many similarities between OSE and FTE compared to other normal cell types, we also were also able to use these profiles to functionally distinguish OSE from FTE. When we compared profiles of HSOC cells, we observed stronger similarities in the regulatory chromatin landscape between FTE and HSOC cell types than between OSE and FTE. Supervised pathway analysis of genes located closest to putative enhancer sites provided additional evidence of signaling pathways that may underlie the neoplastic initiation and progression from normal tissue to HSOC phenotypes. Our results demonstrate the utility of this approach for identifying precursor tissue types for HSOC and other cancers based on the cell type specific chromatin datasets.

Citation Format: Howard C. Shen, Simon Coetzee, Dennis J. Hazelett, Gerhard A. Coetzee, Houtan Noushmehr, Simon A. Gayther. Genome-wide fingerprinting of regulatory chromatin to evaluate the tissue specific origins of high-grade serous ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1380. doi:10.1158/1538-7445.AM2014-1380
Abstract 14: Drug sensitivity of vascularized tumor tissue in vitro
Authors: Ehsan Seema M. , Inoue Masahiro , Waterman Marian L. , Hughes Christopher C. , George Steven C.
Published: May 2015

While there have been significant advances in using 3D culture systems to recapitulate physiological aspects of the tumor microenvironment, few systems are able to capture the full range of authentic, complex in vivo processes such as neovascularization and metastasis. Furthermore, these systems generally utilize established cell lines which compromise the clinical relevance. The goal of this study was to create a 3D in vitro model of patient-specific vascularized tumor tissue that could be used to evaluate the sensitivity to F-fluoruracil (5-FU). Colon cancer cells isolated directly from patient biopsies and endothelial colony forming cell-derived endothelial cells (EC) from cord blood were combined to form multicellular spheroids, and then embedded in a fibrin gel containing normal stromal cells (primary human lung fibroblasts). After 7 days in culture, these “prevascularized tumors” (PVTs) exhibited robust sprouting angiogenesis into the surrounding matrix, and also formed a distinct and contiguous vessel network within the spheroid itself. Network formation and branching of the sprouting vessels was comparable to those generated by co-culture spheroids of EC and cells from the colon cancer cell line SW620. These spheroids in particular demonstrated intravasation of single cells within the lumens of the vessel networks. This phenomenon was enhanced in low oxygen, and abrogated by silencing the transcription factor Slug. Thus, this phenomena is consistent with endothelial-mesenchymal transition. The PVTs have furthermore been used to distinguish the effects of EC on SW620 viability in response to 5-FU. Spheroids composed of EC and SW620 cells demonstrated decreased sensitivity to low dosage 5-FU treatment compared to spheroids composed of SW620 cells only, implicating potential EC-induced drug resistance. Our results demonstrate the utility of a 3D model system of the tumor microenvironment that may be used to study intravasation, microvessel formation, and sensitivity to anti-cancer drugs.

Citation Format: Seema M. Ehsan, Masahiro Inoue, Marian L. Waterman, Christopher CW Hughes, Steven C. George. Drug sensitivity of vascularized tumor tissue in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 14. doi:10.1158/1538-7445.AM2014-14
Abstract 1402: Regulation of Id2 In the proliferation of glioma-initiating neural progenitor cells
Authors: Sullivan Jaclyn , Havrda Matthew , Paolella Brenton , Kettenbach Arminja , Gerber Scott , Israel Mark A.
Published: May 2015

There are more than 14,000 new cases of malignant high grade glioma diagnosed each year in the US, and although surgical resection, radiation, and cytotoxic therapies are available, these current treatment methods are not curative. Inhibitor of DNA Binding 2 (Id2), a helix-loop-helix (HLH) protein, functions as a dominant negative inhibitor of basic helix-loop-helix (bHLH) transcription factors and is highly expressed in glioma. Recent data from our laboratory defines a critical role for Id2 in the pathogenesis of a subset of glioma known as proneural glioma. Id2 protein function has been characterized in neural progenitor cells (NPCs) where it acts to suppress differentiation and enhance cell cycle progression. These are two key characteristics that are shared with glioma-derived stem cells. We have identified three phosphorylation sites on Id2 in proliferating NPCs using mass spectrometry, and prepared altered Id2 molecules that cannot be phosphorylated at each of these sites. Id2-/- NPCs expressing WT and phosphorylation-site-ablated Id2 have been prepared to examine the role of Id2 phosphorylation in mediating its interactions with binding partners and in modulating the rate of proteasome-dependent degradation. We found an increased level of Id2 in NPCs expressing a mutant form of Id2 that cannot by phosphorylated when compared with NPCs expressing WT Id2. Moreover, the level of WT Id2 protein decreases when NPCs exit the cell cycle; however, unphosphorylated Id2 resists this degradation and exhibits a longer half-life. Stable isotope labeling by amino acids in cell culture (SILAC) experiments revealed that unphosphorylated Id2 molecules bind significantly less than WT Id2 to several anaphase promoting complex (APC) subunits. These findings suggested a possible mechanism for phosphorylation-dependent proteasome-mediated degradation of Id2 in NPCs. Understanding the effect of Id2 phosphorylation in modulating steady state levels of this important protein will inform future studies designed to identify pathways of interest in the transformation of NPCs and the malignant progression of NPC-initiated glioma. This work was supported in part by the Theodora B. Betz Foundation.

Citation Format: Jaclyn Sullivan, Matthew Havrda, Brenton Paolella, Arminja Kettenbach, Scott Gerber, Mark A. Israel. Regulation of Id2 In the proliferation of glioma-initiating neural progenitor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1402. doi:10.1158/1538-7445.AM2014-1402
Abstract 1413: RUNX3 plays an oncogenic role in Ewing sarcoma cells
Authors: Bledsoe Krista L. , McGee-Lawrence Meghan E. , Camilleri Emily T. , Oliveira Andre M. , Wijnen Andre J. van , Westendorf Jennifer J.
Published: May 2015

Ewing sarcomas are aggressive, poorly differentiated pediatric tumors of bone and soft tissue. Ewing sarcoma affects one to three people per million every year and is the second most common pediatric bone malignancy after osteosarcoma. Approximately 85% of Ewing sarcomas harbor the EWS-FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS-FLI interacts with numerous lineage-essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS-FLI binds the osteogenic transcription factor RUNX2, and prevents osteoblast differentiation. RUNX3 is highly homologous to RUNX2 and participates in the mesenchymal-derived bone formation. RUNX3 has been described as both a tumor suppressor and an oncogene in different tumor types.

In this study, we investigated the role of RUNX3 in Ewing Sarcoma. RUNX3 was detected in all Ewing sarcoma cells examined (7/7 cell lines, and 4/4 primary tumors), whereas RUNX2 was detected in only 73% of these specimens. Immunoprecipitation experiments revealed that RUNX3 binds to EWS-FLI via its Runt domain, which is >91% identical to the Runt domain in RUNX2. The interaction between RUNX3 and EWS-FLI in cell nuclei was confirmed by immunofluorescence. Moreover, EWS-FLI interactions prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2.

To determine the role of RUNX3 in Ewing sarcoma cell growth, we stably suppressed RUNX3 expression in the Ewing sarcoma cell line A673 with shRNAs. In anchorage independent growth assays in soft agar, RUNX3-deficient A673 cells formed smaller colonies than cells expressing scrambled shRNAs. Consistent with the smaller colonies, RUNX3 suppressed cells had increased expression of the cyclin dependent kinase inhibitor p21. Taken together, these results suggest and oncogenic role for RUNX3 in Ewing sarcoma.

Citation Format: Krista L. Bledsoe, Meghan E. McGee-Lawrence, Emily T. Camilleri, Andre M. Oliveira, Andre J. van Wijnen, Jennifer J. Westendorf. RUNX3 plays an oncogenic role in Ewing sarcoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1413. doi:10.1158/1538-7445.AM2014-1413
Abstract 1415: Prostate cancer: An integrated evaluation of metabolomics, transcriptomics, and proteomics expression data
Authors: Rennefahrt Ulrike , Meyer Hellmuth-A. , Kamlage Beate , Reszka Regina , Schatz Philipp , Stephan Carsten , Jung Klaus , Dietrich Dimo , Kristiansen Glen
Published: May 2015

Background

Metabolite profiling research offers a deeper insight into biochemical changes in cancer metabolism. Moreover the integrated analysis of transcription, metabolomics and proteomics data can improve the understanding of the underlying biological processes.Material and Methods

A set of 254 metabolites was determined by gas chromatography/liquid chromatography-mass spectrometry in matched malignant and non-malignant prostatectomy samples from 95 prostate cancer (PCa) patients. Transcription profiling data obtained from 15 PCa patients by means of Affymetrix U133 arrays was analysed together with public GEO expression data. Expression levels of selected proteins were determined by means of immunohistochemistry and tissue micro array technology in 41 matched frozen tissue samples.

The association with clinicopathological variables and clinical outcome was tested. Transcription and metabolomics data were statistically analysed (ANOVA, Mann-Whitney U test) and significant differentially regulated metabolites/genes/proteins were selected.Results

Differentially regulated metabolites/genes discrimination between malignant and non-malignant tissues was used for network analysis. Enriched pathways which are involved in PCa progression or recurrence such as carbohydrate and fatty acid metabolism were identified. The role of fatty acid metabolism in PCa was analysed in more detail. Several fatty acids such as cerebronic acid, 2-hydroxybehenic acid, tricosanoic acid showed higher concentrations in malignant than in non-malignant tissues. This finding is in concordance to the observed higher mRNA and protein expression level of fatty acid synthase (FASN) in PCa. In contrast to normal prostate tissue, where protein expression level of FASN was correlated to the level of measured metabolites we found in malignant tissues a deregulation of the corresponding pathway. Conclusion

Our integrated analysis of transcription, metabolite and proteomics data confirms and extends the role of several biological pathways which are involved in PCa progression

Citation Format: Ulrike Rennefahrt, Hellmuth-A. Meyer, Beate Kamlage, Regina Reszka, Philipp Schatz, Carsten Stephan, Klaus Jung, Dimo Dietrich, Glen Kristiansen. Prostate cancer: An integrated evaluation of metabolomics, transcriptomics, and proteomics expression data. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1415. doi:10.1158/1538-7445.AM2014-1415
Abstract 1429: Bioluminescent methods for investigating metabolic pathways
Authors: Leippe Donna , Sobol Mary , Vidugiriene Jolanta , Zhou Wenhui , Vidugiris Gediminas , Good Troy , Bernad Laurent , Meisenheimer Poncho , Cali James
Published: May 2015

Cancer cells are dependent on metabolic pathways that have been altered to support unique requirements for cancer cell growth and survival. The central role of these pathways makes them attractive targets for new treatment approaches. Rapid and sensitive assays, amenable to high-throughput screening, are needed to study the enzymes, metabolites and cofactors involved in cancer cell metabolic pathways. We developed a bioluminescent technology to address these needs and monitor changes in the fundamental metabolic co-factors nicotinamide adenine dinucleotides and key metabolites such as lactate.

The bioluminescent method uses a novel proluciferin substrate for the enzyme diaphorase. Luciferin is produced in the presence of NAD(P)H, resulting in a luciferase-generated light signal proportional to the starting NAD(P)H concentration. This is a versatile technology for measuring dinucleotide concentrations, enzyme activity and also metabolite levels. When lactate dehydrogenase is included in the reaction, the cellular metabolite lactate, an indicator of glycolytic rate, can be measured. NAD, NADP, NADH and NADPH can be measured in enzyme reactions or in biological samples, such as cells and tissues, with the use of NAD- and NADP-specific cycling enzymes.

Total NAD+NADH or NADP+NADPH levels in cultured cells were rapidly monitored using a one-reagent addition, in-well plate protocol without the need for cell processing. Additionally, the levels of individual dinucleotides and their ratios were determined from cell and tissue samples using a streamlined in-well acid and base-treatment protocol. The effects of small molecules on cellular dinucleotide levels can be quickly assessed, as demonstrated with FK866, an inhibitor of NAD biosynthesis.

Advantages of the bioluminescent approach over other detection methods include higher sensitivity (LOD ≤ 50 nM), larger maximum signal windows (S/B > 100) and compatibility with automated, high-throughput protocols (Z' > 0.8). By providing these features, the bioluminescent assays should be useful tools for facilitating the study of cancer cell metabolic pathways and the development of therapeutics that target these pathways.

Citation Format: Donna Leippe, Mary Sobol, Jolanta Vidugiriene, Wenhui Zhou, Gediminas Vidugiris, Troy Good, Laurent Bernad, Poncho Meisenheimer, James Cali. Bioluminescent methods for investigating metabolic pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1429. doi:10.1158/1538-7445.AM2014-1429
Abstract 1472: The differential expression of miRNAs in breast cancer cell lines
Authors: Rorie Checo J. , Arrington Brianna L. , Godfrey Sherette S. , McCoy Armeshia S. , Gurley Kashenya M.
Published: May 2015

Breast cancer is a heterogenous disease that is the second leading cause of death amongst women in the United States. Triple negative breast cancer (TNBC) is a very aggressive subtype of breast cancer characterized by its loss of estrogen, progesterone and HER2 receptor expression which limits its targets for effective drug therapies. Micro Ribonucleic Acids (miRNAs) are a class of small, endogenous non-coding ribonucleic acids that seem to play an essential role in gene regulation. Changes in the patterns of miRNA expression profiles are serving as potential biomarkers for tumor diagnosis, prognosis of disease-specific outcomes, and the prediction of therapeutic responses in cancer. We analyzed miRNA array (Agilent) expression profiles in triple negative breast cancer cell lines (HCC70, HCC1806, MDA-MB-157) and non-cancerous (AG11132) African American women breast cell lines. We revealed that the TNBC cells exhibited a different miRNA expression pattern when compared to the normal breast cancer cell line. We analyzed the hybridization data files using standard Microsoft Excel techniques and verified our findings using PARTEK Genomics Suite. We combined the data sets from the TNBC cell lines and compared the miRNA array expression profiles to the normal control cell line AG11132. We revealed that there were significant dysregulation of miRNAs including those of the let-7 family, miR-16, miR-31, and miR-25b-1, miR34a, and miR-95 in the triple negative cells as compared to the normal cells. These miRNAs have been described in previous scientific studies to be associated with tumorigenesis in breast cancer. These studies are initial observations that we will follow up with molecular techniques such as real-time PCR and siRNA to validate the results in order to establish more direct correlations to miRNAs and their roles in triple negative breast cancer.

Citation Format: Checo J. Rorie, Brianna L. Arrington, Sherette S. Godfrey, Armeshia S. McCoy, Kashenya M. Gurley. The differential expression of miRNAs in breast cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1472. doi:10.1158/1538-7445.AM2014-1472
Abstract 1487: Identification of oral carcinoma miRNA signature and control OSCC tumorigenesis through Wnt/β-catenin signaling
Authors: Chang Wei-Min , Hsu Yuan-Ming , Chou Sung-Tau , Shieh Yi-Shing , Hsiao Michael , Hsiao Jenn-Ren , Chang Jang-Yang , Shiah Shine-Gwo
Published: May 2015

Aberrant microRNAs (miRNAs) expressions are often happened in cancer. In order to identify miRNAs altered in oral carcinogenesis, we investigated miRNAs and cDNA expression profiles in 40 pair-wise oral squamous cell carcinoma (OSCC) specimens. Eighty-four miRNAs were differentially expressed in the OSCC specimens compared to the matched tissue. Interestingly, 19 down-regulated miRNAs are clustered in DLK-1/Meg3 imprinted domain of chromosome 14q32.2 region. Loss of 14q32.2-miRNAs expression also raised the 14q32.2-miRNA targets expression in OSCC. We identified one target protein, Wnt-7b, was highly expressed in OSCC and regulated by miR-329 and -410. Wnt-7b increased the Wnt/β-catenin signaling activity and promoted OSCC tumor progression. Furthermore we also identify the regulation mechanism that down-regulated 14q32.2 miRNAs. Hyper-methylation in meg-3 differential methylated region (DMR) was confirmed by bisulfate sequencing. The hyper-methylation of meg-3 DMR was also induced by betel nuts extract exposure. In summary, we identified the expression signature of aberrant miRNAs in OSCC and provide a novel molecular insight how betel quid contribute to oral carcinogenesis through the epigenetic silencing of tumor suppressor miRNAs targeting the Wnt/β-catenin signaling pathway.

Note: This abstract was not presented at the meeting.

Citation Format: Wei-Min Chang, Yuan-Ming Hsu, Sung-Tau Chou, Yi-Shing Shieh, Michael Hsiao, Jenn-Ren Hsiao, Jang-Yang Chang, Shine-Gwo Shiah. Identification of oral carcinoma miRNA signature and control OSCC tumorigenesis through Wnt/β-catenin signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1487. doi:10.1158/1538-7445.AM2014-1487
Abstract 1489: Pri-let-7a-2 rs629367 associated with increased risk and poor survival of gastric cancer in Chinese by up-regulated let-7a expression
Authors: Xu Qian , Yuan Yuan
Published: May 2015

Abstract:Aim:Variant in pri-miRNA could affect miRNA expression and mature process or splicing efficiency, thus altering the hereditary susceptibility and prognosis of cancer. We aimed to assess miRNA-let-7 single nucleotide polymorphisms (SNP) associated with the risk and prognosis of gastric cancer (GC) as predicting biomarkers, and furthermore, its possible mechanisms. Method:We designed a two-stage case-control study to screen four miRNA SNPs (pri-let-7a-2 rs629367 and rs1143770, pri-let-7a-1 rs10739971, pri-let-7f-2 rs17276588) in 107 GC patients, 107 atrophic gastritis (AG), and matched 124 controls using PCR-RFLP. Two promising SNPs (pri-let-7a-2 rs629367 and pri-let-7a-1 rs10739971) were validated in another independent 1949 samples (including 579 GC patients, 649 AG and 721 controls) using Sequenom MassARRAY platform and sequencing. Results: We found that pri-let-7a-2 rs629367 CC variant genotype was associated with increased risks of GC and AG by 1.83-fold and 1.86-fold, respectively. For GC prognosis, patients with rs629367 CC genotype had significantly poorer survival than patients with AA genotype (log-rank P=0.004). We further investigated the let-7a expression levels in serum and found that let-7a expression elevated gradually for rs629367 AA, CA, CC genotype in the AG group (P=0.043). Furthermore, we confirmed these findings in vitro study by overexpressing let-7a carrying pri-let-7a-2 wild-type A or polymorphic-type C allele (P<0.001). Conclusion: pri-let-7a-2 rs629367 CC genotype could increase the risks of GC as well as AG and was also associated with poor survival of GC, which possibly by affecting the mature let-7a expression, and could serve as a predicting biomarker for high-risk and poor prognosis of GC.

Note: This abstract was not presented at the meeting.

Citation Format: Qian Xu, Yuan Yuan. Pri-let-7a-2 rs629367 associated with increased risk and poor survival of gastric cancer in Chinese by up-regulated let-7a expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1489. doi:10.1158/1538-7445.AM2014-1489
Abstract 150: Role of reactive oxygen species in doxorubicin-induced apoptosis of myeloid-derived suppressor cells
Authors: Alizadeh Darya , Katsanis Emmanuel , Larmonier Nicolas
Published: May 2015

The expansion of myeloid-derived suppressor cells (MDSC) in the blood, secondary lymphoid organs and tumor beds in animal cancer models and in patients with many types of cancers participates to the immunosuppressive environment associated with the development of tumors. MDSC frequency correlates with the disease stage and prognosis. MDSC can not only impair innate and adaptive antitumor immune responses, but these cell also contribute to enhance tumor angiogenesis and metastatic potential. The therapeutic targeting of MDSC (depletion,functional inactivation or differentiation into pro-inflammatory cells) promotes the efficacy of immunotherapeutic interventions. Conventional anticancer agents such as docetaxel, gemcitabine or sunitinib have been used to eliminate tumor-induced MDSC. We have observed that the anthracycline doxorubicin can deplete MDSC that expanded in the murine mammary tumor models 4T1 and EMT6 and in the EL4 thymoma model. Doxorubicin-mediated elimination of MDSC is associated with increased T and NK cell activity and the combination of this drug with adoptive T lymphocyte transfer impairs tumor development and metastatic spreading. We here present results indicating that doxorubicin-mediated apoptosis of MDSC may depend on an increased production of reactive oxygen species (ROS) by these cells. MDSC from gp91-/- mice (lacking the gp91phox glycosylated subunit of the NADPH oxidase flavocytochrome b558, responsible for the production of superoxide ion O2·-) were less sensitive to doxorubicin-induced cell death than their wild-type counterparts. Consistently, the effects of doxorubicin on

MDSC were partially impaired in tumor-bearing gp91-/- animals compared to wild-type mice. Interestingly the level of ROS in CD4+ or CD8+ T and NK cells were low and unchanged by doxorubicin administration. These observations may partly explain the selective effects of this chemotherapeutic agent on immunosuppressive MDSC while effector lymphocytes are spared and the subsequent increased in the effector to suppressor cell ratios, resulting in a mitigated immunosuppressive tumor environment.

Citation Format: Darya Alizadeh, Emmanuel Katsanis, Nicolas Larmonier. Role of reactive oxygen species in doxorubicin-induced apoptosis of myeloid-derived suppressor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 150. doi:10.1158/1538-7445.AM2014-150
Abstract 1501: Optimizing small RNAseq of archived serum samples from the Janus Serum Bank - a search for cancer biomarkers
Authors: Rounge Trine B. , Lyle Robert , Lauritzen Marianne , Kjaerheim Kristina , Grotmol Tom , Grimsrud Tom K. , Gislefoss Randi E. , Tretli Steinar , Ursin Giske , Langseth Hilde
Published: May 2015

Introduction

miRNAs are a distinct class of small non-coding RNA molecules that regulate the expression of protein coding genes. They are present and stable in the cell-free body fluids such as plasma, serum and urine. The Janus Serum Bank contains 317 000 serum samples and more than 61 000 incident cancer cases diagnosed during follow-up. It is well-suited for studies of miRNAs as early cancer biomarkers since samples are collected pre-diagnostically.

Aim

To establish a high-throughput sequencing approach for obtaining high quality miRNA expression profiles from archived Janus serum samples stored for up to 41 years. Further aims were to assess the quantity and quality of small RNAs in the samples, and establish guidelines for the optimal sequencing depth.

Methods

Total RNA was extracted from eight pilot samples using Trizol and miRNeasy (Qiagen) columns. Sequencing libraries were made using ScriptMiner (EpiCentre) and the samples were sequenced on one HiSeq (Illumina) lane. After adaptor removal the sequences were aligned unguided to the genome. Reads mapping to mirBASE and Ensembl smallRNA features were counted and sequencing saturation curves were calculated. DESeq was used for differential expression analyses.

Results

High-throughput sequencing results from the Janus samples, with storage times ranging from 13 to 39 years, show that miRNAs are preserved in the samples. High quality miRNA expression profiles, including on average 641 miRNAs per sample, were obtained from our pilot samples. Most of the uniquely mapped reads, on average 84%, are miRNA. We can also identify protein coding RNAs and other groups of small RNA, on average respectively 9% and 6% of the uniquely mapped reads. The preliminary results from sequencing saturation curves, tracking miRNA discovery in each sample as a measure of input reads, have identified a guideline for optimal sequencing depth.

Conclusion and future perspectives

Here we have shown that the stability, quantity and quantity of long-term stored sera from the Janus Serum Bank are sufficient for obtaining high-quality miRNA expression profiles. High-throughput sequencing approach will be used in the search for miRNA expression profiles as early cancer biomarkers for early diagnosis, improved therapy and surveillance of cancer.

Citation Format: Trine B. Rounge, Robert Lyle, Marianne Lauritzen, Kristina Kjaerheim, Tom Grotmol, Tom K. Grimsrud, Randi E. Gislefoss, Steinar Tretli, Giske Ursin, Hilde Langseth. Optimizing small RNAseq of archived serum samples from the Janus Serum Bank - a search for cancer biomarkers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1501. doi:10.1158/1538-7445.AM2014-1501
Abstract 1514: An AC electrokinetic microarray device for isolation of cell circulating free nucleic acid from the blood of cancer patients
Authors: Marciniak Jennifer , Sonnenberg Avery , Rassenti Laura , Ghia Emanuela , Widhopf George , Skowronski Elaine , Manouchehri Sareh , Kipps Thomas J. , Heller Michael J. , Charlot David J. , Krishnan Rajaram
Published: May 2015

Circulating cell-free (ccf) DNA is becoming a biomarker of interest for cancer diagnosis and monitoring. The isolation of ccf-DNA from plasma may be used as a “liquid biopsy” and could replace more invasive tissue biopsies for analysis of cancer-related mutations. Conventional methods for isolation of ccf-DNA from plasma are costly, time-consuming, and require a significant amount of sample processing, which could result in loss of important biomarkers.

An AC electrokinetic microarray device has been developed to reduce the time and cost needed for sample processing. In an earlier study, this microarray device was used to rapidly (15 minutes) isolate ccf-DNA from 25 μL of unprocessed blood from 15 chronic lymphocytic leukemia (CLL) patients to compare PCR and sequencing results with classical ccf-DNA isolation processes and gold standard isolation of DNA directly from lymphocytes. Our objective now is to determine if there is a correlation between the on-chip fluorescence imaging data of the ccf-DNA with the lymphocyte cell counts.

Blood was collected from CLL patients in lithium heparin collection tubes. SYBR Green was added to the CLL blood sample to label the ds-DNA. A 25 μL aliquot of the CLL blood was inserted into the AC electrokinetic microarray device. A 10 kHz sinusoidal waveform was applied at 11 volts peak-to-peak for three minutes without fluid flow. With the field still on, the chip was washed for five minutes with 1x TE at a rate of 100 µL/min to remove other blood components. Once the wash was complete, a fluorescence image was taken for analysis. The white blood cell counts and % lymphocyte data were used to calculate the concentration of lymphocytes in each patient sample, and the fluorescence images were analyzed for average fluorescence in a defined region of interest. The preliminary data shows some correlation between the number of lymphocytes in the patient blood and the fluorescence calculated from the images. It is important to note that while the sample size was small, the data shows promise and calls for a larger study.

Citation Format: Jennifer Marciniak, Avery Sonnenberg, Laura Rassenti, Emanuela Ghia, George Widhopf, Elaine Skowronski, Sareh Manouchehri, Thomas J. Kipps, Michael J. Heller, David J. Charlot, Rajaram Krishnan. An AC electrokinetic microarray device for isolation of cell circulating free nucleic acid from the blood of cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1514. doi:10.1158/1538-7445.AM2014-1514