bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3052: Genomic, transcriptomic, and epigenomic analyses of gastric cancer stem cells
Authors: Kim Seon-Young , Baek Su-Jin , Han Myoung-Eun , Kim Hee-Jin , Kim Jeong-Hwan , Oh Sae-Ock
Published: May 2015

Cancer stem cells are a small population of tumor cells with self-renewal and differentiation capacity, and have been isolated from many types of cancers including stomach. However, the molecular mechanisms underlying the self-renewal and tumorigenic potential of cancer stem cells are poorly understood. To understand the molecular characteristics of gastric cancer stem cells (GCSC), we performed five kinds of experiments including genomic (Exome-seq and Affymetrix SNP 6.0 array), transcriptomic (RNA-seq and small RNA-seq), and epigenomic (MBD-seq) analyses on GCSC, non-gastric cancer stem cells (non-GCSC), and normal tissues from three gastric cancer patients. Among the five data sets, miRNA changes were most significant between GCSC and non-GCSC showing that 39.4% of miRNAs were down-regulated and 1.25% of miRNAs were up-regulated in GCSC compared with non-GCSC. In RNA-seq, 217 (0.93%) genes were up-regulated while 470 (2.02%) genes were down-regulated. Gene-set analysis revealed that development-associated biological processes such as epidermis development and ectoderm development were most enriched terms. In MBD-seq, there were 2.80% of hypermethylated and 2.38% of hypomethylated genes in GCSC compared with non-GCSC. Detailed lists of genes, gene-sets and biological pathways that distinguish GCSC from non-GCSC will be presented and discussed.

Citation Format: Seon-Young Kim, Su-Jin Baek, Myoung-Eun Han, Hee-Jin Kim, Jeong-Hwan Kim, Sae-Ock Oh. Genomic, transcriptomic, and epigenomic analyses of gastric cancer stem 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 3052. doi:10.1158/1538-7445.AM2014-3052
Abstract 3070: Capture, isolation, and mutational analysis of single pancreatic circulating tumor cells using NanoVelcro technology
Authors: Ankeny Jacob S. , Hou Shuang , Lin Millicent , Frias Matthew , OuYang Hank , Song Min , Rochefort Matthew M. , Girgis Mark D. , Tseng Hsian-Rong , Tomlinson James S.
Published: May 2015

Introduction: Circulating tumor cells (CTCs) have been developed as a biomarker in several cancers, but not in pancreatic cancer (PDAC). The available clinical technology for CTC study, CellSearch, suffers from limited ability to perform molecular analysis. We sought to evaluate a novel microfluidic NanoVelcro technology coupled to laser micro-dissection (LMD), for CTC capture and single cell analysis of KRAS mutations to confirm tumor origin of captured CTCs.

Methods: NanoVelcro utilizes a nano-spun polymer fiber, modified with streptavidin, to capture CTCs using biotinylated antibodies. The system was optimized with the CFPAC-1 (EpCAM+, KRAS G12 to V) cell line spiked into healthy donor blood. Patient samples were collected from 2 mL peripheral venous blood and run at the optimized flow rate of 1mL/hr. CTCs were defined by size (≥ 10 µm) and immuno-staining pattern (DAPI+/CK+/CD45-). Using LMD, single cells were isolated and subjected to whole genome amplification followed by PCR of KRAS exon 1 and subsequent Sanger Sequencing.

Results: The NanoVelcro system allowed for detection of mutant KRAS in isolated CFPAC-1 cells. 6 CTCs were then isolated from a known KRAS G12 to V mutant patient and 3/6 (50.0%) captured CTCs were found to have KRAS G12 to V mutations while normal WBCs from the same patient contained wild-type KRAS.

Conclusions: The microfluidic NanoVelcro technology demonstrated capture of pancreatic CTCs and may prove useful in the development of CTCs as a biomarker in PDAC. Application of LMD enabled single cell KRAS mutational analysis confirming cancer origin of isolated CTCs and supporting our current PDAC CTC identification criteria based on immuno-staining. This promising technology, combined with LMD, opens the door for molecular characterization of CTCs, thus providing insight into the biology of metastasis while potentially revealing molecular targets for therapeutics.

Citation Format: Jacob S. Ankeny, Shuang Hou, Millicent Lin, Matthew Frias, Hank OuYang, Min Song, Matthew M. Rochefort, Mark D. Girgis, Hsian-Rong Tseng, James S. Tomlinson. Capture, isolation, and mutational analysis of single pancreatic circulating tumor cells using NanoVelcro technology. [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 3070. doi:10.1158/1538-7445.AM2014-3070
Abstract 3940: Activation of HER2 changes the cetuximab sensitivity in radioresisitant head and neck cancer cells
Authors: Park Jung J. , Kim Sang Yoon , Seo Ji Hyun , Chang Hyo Won , Khalmuratova Roza , Carey Thomas E. , Kim Jin Pyeong , Woo Seung Hoon
Published: May 2015

Background: For patients with recurrent head and neck cancer after radiation therapy (RT), there are limited therapeutic selections. There is great interest in mechanisms of resistance to RT. To investigate these mechanisms, we established RT-resistant clones from head and neck squamous cell carcinoma (HNSCC) cells.

Methods: To develop cell lines with acquired resistance to RT, we utilized the human HNSCC cell line AMC-HN3 and UDSCC-2. We irradiatied clinically relevant cumulative irradiation to cell lines and characterized for the molecular changes in the radioresistant lines relative to the parent cell lines.

Results: A comparative analysis revealed that acquired resistance to RT was associated consistently with the overexpression of HER2 and cross-resistance to cetuximab. To examine the role of HER2, we knocked down HER2 in both radio-resistant and -sensitive parental cells and found an increase in sensitivity to cetuximab. Conversely, inhibition of HER2 in RT-resistant cells was sufficient in resensitizing them to cetuximab in vitro. Further studies indicated that HER2 may diminish sensitivity to cetuximab via activation of the phosphatidyl-inositol-3-kinase (PI3K)/AKT pathway.

Conclusion: Taken together, these findings suggest a central role of HER2 in the development of acquired resistance to RT and prompt consideration to apply HER2 inhibition strategies in future clinical trials of recurrent HNSCC after RT.

Citation Format: Jung Je Park, Sang Yoon Kim, Ji Hyun Seo, Hyo Won Chang, Roza Khalmuratova, Thomas E. Carey, Jin Pyeong Kim, Seung Hoon Woo. Activation of HER2 changes the cetuximab sensitivity in radioresisitant head and neck cancer 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 3940. doi:10.1158/1538-7445.AM2014-3940
Abstract 3946: Wnt/ß-catenin signaling mediates resistance of colorectal cancer cells to chemoradiotherapy
Authors: Emons Georg , Spitzner Melanie , Reineke Sebastian , Möller Janneke , Kendziorra Emil , Beissbarth Tim , Rave-Fraenk Margret , Gaedcke Jochen , Ried Thomas , Ghadimi Michael , Grade Marian
Published: May 2015

Background: Preoperative chemoradiotherapy represents the standard treatment for patients with rectal cancer. However, the clinical response of individual tumors to multimodal treatment is not uniform, and ranges from complete response to complete resistance. Therefore, the identification of novel therapeutic targets whose modification could be harnessed to sensitize a priori resistant tumors is exceedingly important. In this context, we have previously demonstrated that the Wnt transcription factor TCF4 was over-expressed in rectal cancers that were resistant to chemoradiotherapy. The aim of this study was to explore the functional relevance of TCF4 and Wnt/β-catenin signaling for mediating treatment resistance.

Methods: Colorectal cancer cell lines SW837, SW480, and HT-29 were transfected with shRNAs targeting TCF4. After establishing stable single-cell clone (SCC) populations, selected clones were irradiated at 0, 1, 2, 4, 6 and 8 Gy of X-rays. To assess the putative Wnt/β-catenin dependency, β-catenin was inhibited in cancer cell lines using siRNAs, the tankyrase inhibitors XAV939 / JW55 and the porcupine inhibitor LGK 974. Furthermore Wnt/β-catenin signaling activity was stimulated in normal retinal pigment epithelial (RPE) cells through incubation with Wnt3a.

Results: RNAi-mediated silencing of TCF4 led to a significant radiosensitization in SW837 and SW480, two cell lines with a high basal TCF4-activity, whereas no effect was observed in HT-29 a cell line with low activity. Inhibition of β-catenin similarly caused a pronounced sensitization of CRC cells to clinically relevant doses of X-rays. Finally, exogenous stimulation of canonical Wnt signaling with Wnt3a resulted in a significantly decreased sensitivity of RPE cells to radiotherapy, i.e. induced radioresistance.

Conclusion: We have uncovered a novel role of Wnt/β-catenin signaling in mediating resistance to chemoradiotherapy. Moreover, these data suggest that targeting key components of this pathway may represent a potential therapeutic strategy to increase the proportion of patients that respond to chemoradiotherapy, with considerable clinical implications.

Citation Format: Georg Emons, Melanie Spitzner, Sebastian Reineke, Janneke Möller, Emil Kendziorra, Tim Beissbarth, Margret Rave-Fraenk, Jochen Gaedcke, Thomas Ried, Michael Ghadimi, Marian Grade. Wnt/ß-catenin signaling mediates resistance of colorectal cancer cells to chemoradiotherapy. [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 3946. doi:10.1158/1538-7445.AM2014-3946
Abstract 3953: Enhanced radiation reponse in MCF-7 radioresistant cells by targeting Peroxiredoxin II
Authors: Wang Tieli , Diaz Anthony Joseph , Li Jian-Jian , Yen Yun , Tamae Daniel
Published: May 2015

In our previous study, we identified a protein target, Peroxiredoxin II (PrxII) to be overexpressed in radioresistant MCF+FIR3 breast cancer cells and found that its expression and function is associated with breast cancer radiation sensitivity or resistance. Small interference RNA (siRNA) targeting the PrxII gene expression was able to sensitize MCF+FIR3 radioresistant breast cancer cells to ionizing radiation. The major focus of this work was to investigate how the radiation response of MCF+FIR3 radioresistant cells was affected by siRNA that inhibits PrxII gene expression. Our results presented here show that silencing PrxII gene expression increased cellular toxicity by altering cellular thiol status, inhibiting Ca2+ efflux from the cells and perturbing the intracellular Ca2+ homeostasis. By combining radiotherapy and siRNA technology, we hope to develop new therapeutic strategies that may have potential to enhance the efficacy of chemotherapeutic agents due to its targeted property to specific cancer related genes.

Citation Format: Tieli Wang, Anthony Joseph Diaz, Jian-Jian Li, Yun Yen, Daniel Tamae. Enhanced radiation reponse in MCF-7 radioresistant cells by targeting Peroxiredoxin II. [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 3953. doi:10.1158/1538-7445.AM2014-3953
Abstract 3955: 53BP1 facilitates the ATM-dependent phosphorylation of APLF in the DNA damage response
Authors: Fenton Amanda L. , Tran Diana , Koch Christine Anne
Published: May 2015

Aprataxin and polynucleotide kinase-like factor (APLF) is a DNA repair factor which facilitates DNA repair in part through interactions with its forkhead-associated (FHA) domain. The APLF FHA domain mediates interactions with specific threonine phosphorylated epitopes via a functionally conserved and essential arginine residue (Arg-27). In the context of DNA double-strand break (DSB) repair, substitution of APLF amino acid residue Arg-27 to alanine abolishes interactions with the DSB repair scaffold protein XRCC4, and impairs DSB repair. APLF is also a component of the ATM-dependent DNA damage response (DDR), which also includes 53BP1, and undergoes ionizing-radiation induced phosphorylation at Ser-116 facilitating its role in DSB repair. Here we demonstrate that the APLF FHA domain is essential for Ser-116 phosphorylation and for the association with 53BP1, which collectively enhance the accumulation of APLF at sites of DNA damage. The level of 53BP1 expression, but not XRCC4, was found to correlate with the phosphorylation of APLF by ATM, and disruption of the APLF-53BP1 interaction was associated with impaired cell survival following DNA damage. These results suggest that the APLF FHA domain directs interactions with multiple partners in the DNA damage response, and that 53BP1 facilitates the ATM-dependent phosphorylation of APLF.

Citation Format: Amanda L. Fenton, Diana Tran, Christine Anne Koch. 53BP1 facilitates the ATM-dependent phosphorylation of APLF in the DNA damage response. [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 3955. doi:10.1158/1538-7445.AM2014-3955
Abstract 3966: PLK1 regulates PAX3-FOXO1 stability and its inhibition mediates regression of alveolar rhabdomyosarcoma xenograft tumors
Authors: Thalhammer Verena , Herrero-Martin David , Hecker Regina , Laubscher Dominik , Lopez-Garcia Laura , Wachtel Marco , Bode Peter , Schäfer Beat
Published: May 2015

Oncogenic addiction provides an opportunity to develop new treatment options, especially for childhood cancers. Pediatric tumors contain a lower number of oncogenic mutations compared to most adult cancers, suggesting stronger dependency on individual oncogenes, such as chimeric transcription factors that have the ability to control multiple oncogenic pathways. Taking advantage of this addiction, targeting of oncogenic transcription factors becomes a new powerful strategy for therapy of translocation positive pediatric tumors like alveolar rhabdomyosarcoma (aRMS), which is characterized by a very dismal prognosis.

As transcription factors are considered to be difficult to target, we developed an approach to screen for druggable upstream regulators like kinases. In two parallel activity reporter assays, using a kinome siRNA library and a small molecule library, we identified PLK1 to contribute to the activity of PAX3-FOXO1, the oncogenic transcription factor specific to aRMS. Mechanistically, we were able to demonstrate a direct interaction of the two proteins by co-immunoprecipitation. PLK1 silencing or inhibition increased degradation of PAX3-FOXO1, which suggests a stabilization of PAX3-FOXO1 due to phosphorylation by PLK1. Candidate phosphorylation sites were identified by in vitro kinase assays and mass spectrometric analysis and site-specific mutagenesis is currently utilized to validate the impact of these sites on fusion protein turnover. To highlight PLK1 as therapeutic target in aRMS, in vivo treatment studies using the PLK1 inhibitor BI 2536 resulted in complete tumor regression in two aRMS xenograft mouse models. Finally, tissue microarray analysis of human aRMS tumor biopsies showed PLK1 to be overexpressed and predictive of overall survival.

Hence, our preclinical studies validate PLK1 as highly relevant drug target in alveolar rhabdomyosarcoma, similar to recent findings in other pediatric tumors. Our data suggests that PLK1 inhibitors warrant further investigations to evaluate the potential to be translated into a clinical setting.

Citation Format: Verena Thalhammer, David Herrero-Martin, Regina Hecker, Dominik Laubscher, Laura Lopez-Garcia, Marco Wachtel, Peter Bode, Beat Schäfer. PLK1 regulates PAX3-FOXO1 stability and its inhibition mediates regression of alveolar rhabdomyosarcoma xenograft tumors. [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 3966. doi:10.1158/1538-7445.AM2014-3966
Abstract 3980: Pre-clinical evaluation of a novel DNA crosslinking agent, Ureidomustin (BO-1055) in pediatric sarcomas
Authors: Ambati Srikanth R. , Wong Elissa W. , Pera Benet , Peguero Elizabeth , Lopes Eloisi Caldas , Chen Jin-Jer , Shieh Jae-Hung , Su Tsann-Long , Moore Malcolm A.
Published: May 2015

DNA crosslinking agents continue to be an important part of chemotherapy for pediatric sarcomas. We tested potent analogues of phenyl N-mustard-benzene conjugates in several tumor models. In this work, we compared the efficacy of a novel water-soluble DNA alkylating agent, Ureidomustin (BO-1055) in Ewing sarcoma, rhabdomyosarcoma, osteosarcoma, desmoplastic small round cell tumor (DSRCT) cell lines and early cultures derived from primary patient samples. We tested its toxicity in normal human cells in vitro including the proliferation of mesenchymal stromal cells, bone marrow-derived endothelial cells and purified cord blood CD34+ cells in colony-forming assay (for hematopoietic progenitor cells) and in cobblestone area forming assay (for hematopoietic stem cells). We performed cell viability, cytotoxicity and caspase activation assays in a dose-dependent and time-dependent manner. We also performed apoptosis, cell cycle analyses using flow cytometry, immunoblot analysis and in vivo experiments in nude mice using Ewing sarcoma (A673) cell line transduced with GFP/luciferase for bioimaging. We evaluated cardiotoxicity in the HL-1 cardiomyocyte line in vitro and noted a 227 fold less cytotoxicity using BO-1055 compared to Doxorubicin. Based on MTD testing in nude mice, we used 30mg/kg q2d for 5 doses after tumors reached 75-100mm3 and were detected on luciferase bioimaging. Our results indicated that BO-1055 has a significant therapeutic window in its activity (mean IC50±SD μM) for rhabdomyosarcoma (0.16±0.11), Ewing sarcoma (0.48±0.29) and DSRCT (3.07±0.94) but not for osteosarcoma (IC50>10μM). The mean IC50 in benign cells was >10μM. Treatment with BO-1055 resulted in G2 arrest and activation of caspases 3 and 7 in a dose dependent manner. A673 xenografts were significantly inhibited by BO-1055 compared to the control mice injected with vehicle, based on mean fluorescence intensity and size of the tumors. Our results suggest that BO-1055 has potent activity against Ewing sarcoma and rhabdomyosarcoma and presents a strong rationale for further development as a therapeutic agent.

Citation Format: Srikanth R. Ambati, Elissa WP Wong, Benet Pera, Elizabeth Peguero, Eloisi Caldas Lopes, Jin-Jer Chen, Jae-Hung Shieh, Tsann-Long Su, Malcolm AS Moore. Pre-clinical evaluation of a novel DNA crosslinking agent, Ureidomustin (BO-1055) in pediatric sarcomas. [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 3980. doi:10.1158/1538-7445.AM2014-3980
Abstract 399: Establishment of hypomethylating agent-resistant cell lines using a cell line from a patient with acute myeloid leukemia evolved from myelodysplastic syndrome (MOLM-13)
Authors: Choi Dae R. , Hur Eun-Hye , Moon Ju Hyun , Goo Bon-Kwan , Choi Yunsuk , Lee Do Young , Ro Seonggu , Cho Joong Myung , Lee Je-Hwan
Published: May 2015

Myelodysplastic syndrome (MDS) is a clonal disorder of the hematopoietic cells occurring in elderly patients in a majority of instances. They are defined by ineffective hematopoiesis with peripheral blood cytopenias and by the risk of evolution to acute myeloid leukemia (AML). Hypomethylating agents (HMA), 5-azacytidine (AZA) and 2-deoxy-5-azacytidine (DAC), have demonstrated clinical activities with cytologic and cytogenetic responses in patients with MDS, but potential problems of both agents include the development of intrinsic and acquired resistance.

HMA-resistant patients have very poor prognosis, and this cohort of patients constitutes an important area of research. To understand mechanisms of HMA-resistance and overcome the resistance, we tried to establish HMA-resistant cells using MOLM-13, which is a cell line from a patient with AML evolved from MDS.

Original MOLM-13 cell line was sensitive to both AZA and DAC in vitro. MOLM-13 cells were exposed continuously to increasing concetrations of AZA or DAC and we could establish an AZA-resistant cell line (MOLM/AZA-1) and a DAC-resistant cell line (MOLM/DAC-5). The IC50 of AZA in MOLM/AZA-1 was 96.26 µM compared to 0.11 µM of the parent cell line; 800-fold increase in resistance to AZA. The IC50 of DAC in MOLM/DAC-5 was 207.7 µM compared to 0.07 µM of the parent cell line; 2000-fold increase in resistance to DAC. We treated th AZA- and DAC-resistant cells with vorinostat (SAHA) and a novel agent, CG200745 to test whether the histone deacetylase (HDAC) inhibitors could overcome the HMA-resistance. Cell viability assays showed that HMA-resistant cells were sensitive to both HDAC inhibitors, which dramatically decreased anti-apoptotic proteins (XIAP, MCL-1 and BCL-2) and increased PARP cleavages in the HMA-resistant cell lines. In conclusion, we successfully established established AZA- and DAC-resistant cell lines from an AML/MDS cell line, MOLM-13, and found that HDAC inhibitors might be promising in overcoming HMA-resistance. Further studies with the HMA-resistant cell lines are ongoing for elucidation of resistance mechanisms and possible role of other novel agents to overcome the HMA-resistance.

Citation Format: Dae Ro Choi, Eun-Hye Hur, Ju Hyun Moon, Bon-Kwan Goo, Yunsuk Choi, Do Young Lee, Seonggu Ro, Joong Myung Cho, Je-Hwan Lee. Establishment of hypomethylating agent-resistant cell lines using a cell line from a patient with acute myeloid leukemia evolved from myelodysplastic syndrome (MOLM-13). [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 399. doi:10.1158/1538-7445.AM2014-399
Abstract 3993: A microarray-based gene expression analysis identified diagnostic biomarkers for unknown primary cancer
Authors: Fujita Yoshihiko , Kurahashi Issei , Kurata Takayasu , Koh Yasuhiro , Sakai Kazuko , Nakagawa Kazuhiko , Nishio Kazuto
Published: May 2015

Background: Patients with cancer of unknown primary (CUP) present with metastatic disease for which the primary site cannot be found. Clinically, CUPs exhibit common characteristics, such as rapid progression and early dissemination, with a silent primary tumor that may become involuted and undetectable. Existence of such common properties prompts us to hypothesize that there may be potential biological markers that elucidate CUP as a whole.

Methods: Tumor mRNA samples from 60 patients with CUP were measured for the expression of ∼22,000 genes using the Affymetrix U133A Plus 2.0 GeneChip and analyzed by applying normalization and classification algorithms to the gene expression data. The similarity of each tumor specimen's gene expression pattern is then compared to the gene-expression profiles specific to non-CUP groups (containing tumors from 24 known primary sites) that were constructed using publicly available raw microarray datasets. The t-tests were performed to compare the CUP with non-CUP groups and the top 59 CUP specific genes with the highest fold change were selected (p-value < 0.001).

Results: This study enabled the identification of genes that exhibited a unique expression pattern in CUP. As a high metastasis potential and vulnerability to apoptosis would explain the properties of CUP well, we first searched for genes related to metastasis and apoptosis and found 14 genes among 44 that were up-regulated by more than 2.5-fold in the CUP samples. Some of these genes were associated with the epithelial-to-mesenchymal transition (EMT), a function that has been increasingly recognized as a key step in cancer metastasis. We also identified 6 genes for ribosomal proteins among 44 up-regulated genes, two of which (RPS7 and RPL11) were known to be involved in the Mdm2 - p53 pathway.

Conclusions: The protein products of the up-regulated and down-regulated genes identified in this study suggest a biological attribute of CUP and, therefore, may become clinically useful biomarkers for CUP.

Citation Format: Yoshihiko Fujita, Issei Kurahashi, Takayasu Kurata, Yasuhiro Koh, Kazuko Sakai, Kazuhiko Nakagawa, Kazuto Nishio. A microarray-based gene expression analysis identified diagnostic biomarkers for unknown primary 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 3993. doi:10.1158/1538-7445.AM2014-3993
Abstract 3999: Characterization of the limiting steps in the melanoma metastatic cascade
Authors: Piskounova Elena , Morrison Sean
Published: May 2015

Metastasis is a complex multistep process: cancer cells have to detach from the bulk of the primary tumor, intravasate into the blood, survive anoikis and high shear forces in the blood stream while existing as single cells, extravasate into a distal site, and finally proliferate to form metastatic nodules. Each of these steps requires distinctive capabilities, which must either be intrinsic or gained.

We have used a melanoma xenograft model to define three major limiting steps of the metastatic cascade: intravasation into the blood, survival in circulation/extravasation into organs, and proliferation at distal sites. In our model, melanoma tumors that have previously been shown to efficiently metastasize to distal organs when grown subcutaneously (efficient metastasizers), are also capable of overcoming each of these three bottlenecks of metastasis. On the contrary, cells that are not able to metastasize when grown subcutaneously (inefficient metastasizers), are also inefficient at each of these limiting steps. This suggests that there are significant intrinsic differences between these two classes of tumors that determine the capacity of melanoma cells to grow at each step of the metastatic cascade.

To gain mechanistic insight into these differences, we have analyzed genomic mutations, gene expression and metabolomic profiles, comparing human melanomas with varying metastatic potential. Using our xenograft model we are currently investigating the role of select pathways in driving melanoma metastasis, which will ultimately lead to improved therapeutic options for patients with aggressive cancers.

Citation Format: Elena Piskounova, Sean Morrison. Characterization of the limiting steps in the melanoma metastatic cascade. [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 3999. doi:10.1158/1538-7445.AM2014-3999
Abstract 4002: Exploring the miRNA and proteomic contents of exosomes isolated from rhabdomyosarcoma tumor cells to understand their paracrine signaling
Authors: Ghayad Sandra E. , Ghamloush Farah , Saab Raya
Published: May 2015

Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, with two distinct subtypes, alveolar (ARMS) and embryonal (ERMS) histologies. The alveolar subtype is characterized by a specific translocation PAX-FOXO, the protein product of which is thought to contribute to its aggressive and metastatic behaviour.

Exosomes are small membranous vesicles (30-100 nm in diameter) secreted into body fluids by multiple cell types, including tumor cells. Tumor exosomes contain intact and functional protein and microRNA that can alter the cellular environment to favor tumor growth.

We hypothesize that the PAX-FOXO fusion protein results in specific effects on exosome cargo and biology, contributing to the invasive and metastatic potential of ARMS cells. Thus proteomic and molecular profiling of their exosomes may increase our understanding of the role of exosomes in sarcoma progression and may lead to discovery of useful biomarkers.

In the present study, we used miRNA array profiling to identify interesting exosomal miRNA expressed commonly in two different ARMS cell lines. We also used proteomic analysis and discovered differentially expressed ARMS exosomal proteins. Importantly, functional studies revealed that normal fibroblasts acquired invasion and migration ability through molecules transported in ARMS cell-derived exosomes.

Our results indicate that ARMS-derived exosomes have unique gene expression signatures,miRNA and proteomics profiles which will help our understanding of sarcoma biogenesis and that could be used as biomarkers for early diagnosis of ARMS, and targets for therapeutic intervention.

Citation Format: Sandra E. Ghayad, Farah Ghamloush, Raya Saab. Exploring the miRNA and proteomic contents of exosomes isolated from rhabdomyosarcoma tumor cells to understand their paracrine 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 4002. doi:10.1158/1538-7445.AM2014-4002
Abstract 4011: Single cell transcriptomic analysis identified a potential dormant signature in prostate cancer disseminated tumor cells
Authors: Lam Hung-Ming , Chéry Lisly , Coleman Ilsa , Lakely Bryce , Larson Sandy , Coleman Roger , Aguirre-Ghiso Julio , Xia Jing , Gulati Roman , Nelson Peter S. , Montgomery Bruce , Lange Paul H. , Snyder Linda A. , Vessella Robert L. , Morrissey Colm
Published: May 2015

Prostate cancer (PCa) disseminates before radical prostatectomy and can remain in the bone marrow for a prolonged period of time (>10 years) until lethal metastasis develops. These cells are referred to as disseminated tumor cells (DTC). Dormant DTC are resistant to current chemotherapy targeting cell division, therefore understanding the nature of DTC will allow the development of novel drug target to prevent overt cancer metastases.

DTC were isolated from the bone marrow aspirates of PCa patients with no evidence of disease (NED, undetectable PSA level after 7-18 years after radical prostatectomy) or advanced diseases (ADV, disease progression after treatment or existing distant metastases). Eighty-five EpCAM+/CD45- individual cells were subjected to microarray gene expression analyses and two populations of cells were identified: erythroid progenitor-like and prostate epithelial cells. We utilized a dual signature method to identify EpCAM+/CD45- cells that are of prostatic origin. Comparison among prostate DTC showed that the DTC population within each patient was heterogeneous. Importantly, comparison between DTC from NED and ADV patients revealed that DTC from NED patients were enriched in a dormancy-associated signature identified in the head and neck squamous cell carcinoma, supporting the dormant nature of DTC from NED patients. Global clustering analysis and Ingenuity Pathway Analysis further identified a potential PCa dormancy signature, and this signature is significantly suppressed in a subpopulation of DTC isolated from ADV patients.

We reported a single cell transcriptomic analysis to reveal for the first time clinically heterogeneous DTC population in PCa patients and a dual signature method to identify cells of prostatic origin from erythroid-progenitor cells that harbored the same epithelial (EpCAM) surface marker in the bone marrow. The proposed gene signature associated with PCa dormancy may allow development of possible biomarkers to predict prognosis and therapeutic targets to promote PCa dormancy or prevent dormancy escape.

Citation Format: Hung-Ming Lam, Lisly Chéry, Ilsa Coleman, Bryce Lakely, Sandy Larson, Roger Coleman, Julio Aguirre-Ghiso, Jing Xia, Roman Gulati, Peter S. Nelson, Bruce Montgomery, Paul H. Lange, Linda A. Snyder, Robert L. Vessella, Colm Morrissey. Single cell transcriptomic analysis identified a potential dormant signature in prostate cancer disseminated tumor 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 4011. doi:10.1158/1538-7445.AM2014-4011
Abstract 4018: Development and analytical validation of a novel real-time multiplex RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR mRNA expression in circulating tumor cells of breast cancer patients
Authors: Strati Areti D. , Lianidou Evi S.
Published: May 2015

Introduction: Circulating Tumor Cells (CTCs) represent an important biological link in the spread of breast cancer from primary to metastatic disease. CTCs have already been established as strong predictors of prognosis in patients with metastatic breast cancer. Triple-negative breast cancers have a more aggressive clinical outcome than other forms of breast cancer. The aim of our work was to develop a quantitative real-time multiplex RT-qPCR assay for studying the expression of ER, PR, HER-2 and EGFR in CTC of breast cancer patients.

Materials and Methods: A quadruplex quantitative real time RT-qPCR assay for ER, PR, HER-2 and EGFR was developed based on the de novo in-silico design of eight primers and four dual hybridization probes. All primers and probes were designed to avoid cross-reactions and primer dimer formation and to match the assay conditions, such as amplicon sizes, and melting temperatures. The specificity of all primer and hybridization probe sequences was first tested by homology searches in the nucleotide database (NCBI, nucleotide BLAST). Each probe set included a 3′-fluorescein (F) donor probe and a 5′- LC acceptor probe that was different for each gene set: ER at 610nm, PR at 640nm, HER-2 at 670nm, EGFR at 705nm. A color compensation test was performed by using pure dye spectra so that spectral overlap between dyes was corrected. The LightCycler 2.0 platform, (Roche, Diagnostics) was used since it allows detection of six multiple reporter dyes in the same capillary. The assay was designed, so that only 1 μL of cDNA is used and the total reaction volume is 10 μL.

Results: The assay sensitivity was evaluated by performing spiking experiments. For this reason known numbers of MCF7 and SKBR-3 cells, as well as external quantification calibrators ranging from 105 copies/μL to 10 copies/μL, were used. The specificity was evaluated by carefully designed cross reaction studies, performed for each gene target in the presence of all other targets. Our assay, under optimized conditions has shown a very high specificity for each analyte.

Conclusions: We report for the first time a highly sensitive, specific and reproducible quantitative multiplex real-time RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR. The assay is currently validated in CTC isolated from a large number of breast cancer patients.

Citation Format: Areti D. Strati, Evi S. Lianidou. Development and analytical validation of a novel real-time multiplex RT-qPCR assay for the simultaneous quantification of ER, PR, HER-2 and EGFR mRNA expression in circulating tumor cells of breast 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 4018. doi:10.1158/1538-7445.AM2014-4018
Abstract 4020: Exploring target gene(s) within chromosome 19-amplification detected in a subclone from metastatic tumors in mouse transplantable OSCC
Authors: Muramatsu Tomoki , Kozaki Ken-ichi , Imoto Seiya , Yamaguchi Rui , Tsuda Hitoshi , Kawano Tatsuyuki , Miyano Satoru , Inazawa Johji
Published: May 2015

Cancer metastasis is caused by a highly complex molecular and cellular mechanism. Although this mechanism has been gradually revealed, there are few genes found as useful diagnostic markers or molecular targets for clinical application so far. In this study, we attempted to establish highly metastatic cells by in vivo selection for identifying metastasis-related genes with an oral squamous cell carcinoma cell line (HOC313-parent). As a result, we established a highly metastatic subline (HOC313-LM) derived from a metastatic site of axillary lymph node. To explore metastasis-related genes and signaling pathways, we performed integrative omics including several genomic and epigenomic approaches combined with systems biology. Since the amplification of chromosome 19 was identified in HOC313-LM by array CGH, we focused on this amplicon containing 44 genes. Among them, one gene, which was essential for the posttranslational modification, was associated with tumor formation and metastasis in vivo via the Rho signaling pathway. Indeed, activation of the Rho signaling pathway emerged in HOC313-LM by our systems biology approach using the gene expression data sets. In addition, the expression pattern of this gene correlated with poor disease-free survival and distant metastasis in patients with esophageal squamous cell carcinoma. Then, we performed therapeutic experiments using a known inhibitor of this gene, and found that tumor growth was inhibited by this compound. Taken together, our findings suggested that this gene might serve as a novel molecular target for anti-cancer therapy.

Citation Format: Tomoki Muramatsu, Ken-ichi Kozaki, Seiya Imoto, Rui Yamaguchi, Hitoshi Tsuda, Tatsuyuki Kawano, Satoru Miyano, Johji Inazawa. Exploring target gene(s) within chromosome 19-amplification detected in a subclone from metastatic tumors in mouse transplantable OSCC. [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 4020. doi:10.1158/1538-7445.AM2014-4020
Abstract 4026: Attenuation of pancreatic cancer cell migration and invasion through a targeted inhibition of the Rac GEF Vav1
Authors: Razidlo Gina L. , Magnine Christopher , Sletten Arthur C. , Hurley Rachel M. , McNiven Mark A.
Published: May 2015

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of human cancer, due in large part to a high incidence of metastasis and resistance to conventional chemotherapies. As such, new therapies are needed to halt the metastatic process. The proto-oncogene Vav1 is ectopically expressed in over half of human pancreatic cancers, and its expression correlates with a poor prognosis in humans and promotes survival and transformation in isolated tumor cells. We have found that Vav1, through its GEF activity towards Rac1, also potently induces migration by pancreatic tumor cells. In addition, Vav1 also signals through Cdc42 to drive the formation of invadopodia and matrix degradation, which also promotes tumor cell invasion. Importantly, the activation of Vav1 circumvents the requirement for active Src in this process. Because Vav1 is ectopically expressed in pancreatic cancers and is a potent regulator of tumor cell survival, proliferation, and invasive migration, it could provide a specific therapeutic target for these tumors. Recently, the anti-inflammatory agent azathioprine was described as an inhibitor of Vav1/Rac1 activity in hematopoietic cells. Therefore, we hypothesized that azathioprine could also inhibit the Vav1-driven processes of proliferation and invasion in pancreatic tumor cells. Indeed, we have determined that azathioprine treatment of cultured human PDAC cells reduces viability and proliferation. Further, similar to siRNA-mediated depletion of Vav1, azathioprine treatment reduces cell migration, as well as the matrix degradation required for invasion. Vav1-expressing tumor cells were more sensitive to azathioprine than Vav1-negative cells, supporting the premise that the inhibitory effects of azathioprine are mediated through Vav1. Finally, azathioprine also reduces metastasis in a xenograft model of pancreatic cancer. Taken together, these data uncover a role for aberrantly expressed Vav1 in regulation of the cytoskeletal machinery required for migration and invasion, and in the promotion and metastasis of pancreatic cancer. Even with the expression of multiple Rho family GEFs, tumor cells that upregulate Vav1 have become dependent upon this exchange factor, implicating Vav1 as a potent node for therapeutic intervention. Supported by R01 CA104125 and R03 CA155778 to M.A.M.

Citation Format: Gina L. Razidlo, Christopher Magnine, Arthur C. Sletten, Rachel M. Hurley, Mark A. McNiven. Attenuation of pancreatic cancer cell migration and invasion through a targeted inhibition of the Rac GEF Vav1. [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 4026. doi:10.1158/1538-7445.AM2014-4026
Abstract 4034: The anti-peritoneal metastasis properties of Yangzheng Xiaoji, the potential role of hyaluronan and CD44
Authors: Jiang Wen G. , Jia Yongning , Ye Lin , Ji Ke , Gao Yong , Wei Cong , Wu Yiling , Topley Nick , Kift-Morgan Anna , Ji Jiafu
Published: May 2015

Introduction. Peritoneal metastasis is a frequently occurring complication patients with gastrointestinal, pancreatic, ovarian cancers and is a condition difficult to manage, clinically, due largely to the lack of effective treatment. In the present study, we report an accidental and novel discovery that the extract of Yangzheng Xiaoji (YZXJ), a traditional medical formula used in cancer treatment had an anti-peritoneal metastasis function.

Materials and methods. Human primary cultured peritoneal mesothelial cells were used as an in vitro cellular model. Human gastric, colorectal, pancreatic, ovarian and prostate cancer cells were used in the tests. Tumour-mesothelial interaction, invasion and migration were assessed with YZXJ. Athymic nude mice peritoneal metastasis model was used to evaluate the effects in vivo.

Results. Soluble YZXJ was firstly extracted in a highly reproducible manner. YZXJ extract (DME25) had a negletable toxicity to cancer cells and mesothelial cells. However, it profoundly inhibited the tumour-mesothelial adhesions in the type of tumour cells tested with pancreatic and colorectal cancer cells amongst the most sensitive ones to YZXJ (p<0.001), a phenomenon seen with a number of cell based assays. The inhibitory effect was also seen with cellular migration. A similar pattern of inhibition was seen on tumour adhesion to hyaluronan (HA) coated surface. It was further revealed that YZXJ and an hyaluronan inhibitor and anti-CD44 antibodies had additive effects with YZXJ on tumour-mesothelial interactions. YZXJ extract had an inhibitory effect on the phosphorylation of SRC kinase in cancer cells and in mesothelial cells as shown by Western blotting and immunofluorescence microscopy. In the peritoneal metastasis in vivo model, it was demonstrated that intraperitoneal administration of YZXJ significantly reduced the formation of metastatic nodules (p=0.035, control vs YZXJ treatment). This was seen with little side effects. We have also shown that hyaluronan inhibitor strengthened the inhibitory effect exerted by YZXJ.

Conclusion. Yangzheng Xiaoji has an potent inhibitory effect on tumour-mesothelial interactions of a variety of tumour types and also markedly reduces the peritoneal metastasis in vivo. This effect is likely to be achieved by its effect on the SRC signalling pathway. Yangzheng Xiaoji presents a new way of treating peritoneal metastasis.

Citation Format: Wen G. Jiang, Yongning Jia, Lin Ye, Ke Ji, Yong Gao, Cong Wei, Yiling Wu, Nick Topley, Anna Kift-Morgan, Jiafu Ji. The anti-peritoneal metastasis properties of Yangzheng Xiaoji, the potential role of hyaluronan and CD44. [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 4034. doi:10.1158/1538-7445.AM2014-4034
Abstract 4037: Combining tyrosine kinase inhibitors and isoflavones to target metastatic activity in prostate cancer cells
Authors: Rice Lori P. , Pampo Christine , Lepler Sharon , Siemann Dietmar W.
Published: May 2015

Background: Efforts to impede early steps in the metastatic cascade have led to the development of promising small molecule targeting agents. The purpose of this study was to evaluate the use of Src tyrosine kinase inhibitors known to interfere with cell migration and invasion, in combination with a cytostatic soy isoflavone extract (ISFs) on metastatic activity in prostate cancer cells. Methods: PC-3ML cells (a gift from A. Fatatis, Drexel University), and LNCaP cells treated with a Src inhibitor, ISFs, or a combination, were evaluated for clonogenic cell survival, cell cycle progression, and transwell migration and invasion activity. Results: Src inhibitors and ISFs induced an accumulation of cells in the G1 and G2/M phases of the cell cycle, respectively, with little change in cell viability or survival. Invasion studies with PC-3 ML and LNCaP cells suggest that ISFs can reduce metastatic activity by 39% and 35%, respectively. In addition, ISFs enhance the effectiveness of the Src inhibitors to reduce invasiveness. This may indicate that including ISFs in treatment regimens may allow a lower dose of the targeting agent to be used to achieve optimal response and decrease toxicity. Conclusions: In vitro studies suggested that combining a Src inhibitor and ISFs resulted in greater inhibition of metastatic activity than either alone.

Citation Format: Lori P. Rice, Christine Pampo, Sharon Lepler, Dietmar W. Siemann. Combining tyrosine kinase inhibitors and isoflavones to target metastatic activity in prostate cancer 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 4037. doi:10.1158/1538-7445.AM2014-4037
Abstract 404: Function of Brg1 chromatin remodeling factor in sonic hedgehog-dependent medulloblastoma development
Authors: Shi Xuanming , Zhang Zilai , Wang Qiu , Wu Jiang
Published: May 2015

Medulloblastoma is the most common malignant pediatric brain tumor. Overactive Shh signaling in cerebellum granule neuron precursors (CGNPs) is the leading cause of the childhood medulloblastoma (Shh-subtype). Previously we have shown that chromatin remodeler Brg1 is required for Shh target gene expression, and Brg1 deletion reduced CGNP proliferation in developing cerebellum. Current study focuses on the function of Brg1 in mouse model of Shh-subtype medulloblastoma. In CGNP cultured from SmoM2 transgenic mice where Shh pathway is constitutively active, we found Brg1 is required for CGNP mitogenic target gene expression and proliferation. In SmoM2 medulloblastoma cultures, we observed that tumor cell growth was inhibited by conditional knockout of Brg1. In subcutaneous transplantation, we found that tumors were significantly shrunk upon induction of Brg1 deletion. Detailed analysis indicated that Shh-dependent mitogenic target genes decreased by loss of Brg1. Further evidences showed that medulloblastoma cell proliferation was significant inhibited by conditional knockout of Brg1. Effect of Brg1 on the chromatin environment of target genes during medulloblastoma development will be discussed. Our study will provide insights to the epigenetic mechanism of Shh-dependent tumor development and new therapeutic targets.

Citation Format: Xuanming Shi, Zilai Zhang, Qiu Wang, Jiang Wu. Function of Brg1 chromatin remodeling factor in sonic hedgehog-dependent medulloblastoma development. [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 404. doi:10.1158/1538-7445.AM2014-404