bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3511: Sustained adrenergic signaling promotes cervical cancer progression
Authors: Sadaoui Nouara C. , Armaiz-Pena Guillermo N. , Nagaraja Archana S. , Rupaimoole Rajesha , Previs Rebecca A. , Dalton Heather J. , Lingegowda Mangala S. , Ramondetta Lois M. , Sood Anil K. , Lutgendorf Susan K. , Cole Steve W.
Published: May 2015

Objective: Chronic stress and sustained adrenergic signaling promote tumor progression. The underlying mechanisms behind this process are not well understood. We examined the effects of sustained adrenergic signaling on cervical cancer progression through increased expression of HPV oncogenes, E6 and E7.

Methods: Beta-adrenergic receptor (ADRB) positive cervical cancer cell lines (CaSki and SiHa) were treated with norepinephrine (NE) or isoproterenol (ISO) to analyze intracellular responses. Migration, invasion and anoikis assays were performed to elucidate the resultant biological effect of NE and ISO. ADRB expression levels were examined from cervical cancer patient tumor samples.

Results: ADRB expression was determined for cervical cell lines (CaSki, SiHa, and C33A) using qRT-PCR. CaSki and SiHa cells express ADRB1, ADRB2, and ABRB3. Cells exposed to NE for 30 min showed elevated cyclic AMP activity. After treatment with NE or ISO, mRNA levels from HPV oncogenes, E6 and E7, were significantly elevated in the SiHa and CaSki cells. NE exposure resulted in a significant increase in invasion and migration of cervical cancer cells, while E6 siRNA abrogated these effects. After 48 hours of NE or ISO exposure, CaSki cells showed a 30% reduction in anoikis. Among 166 tumor samples evaluated from cervical cancer patients, 85% had increased ADRB1 expression and 61% had increased ADRB2 expression. Tumor stage or grade was not related to ADRB expression. ADRB1 expression was not correlated with patient survival outcomes (p = 0.86); however, patients with high ADRB2 expressed had decreased overall survival (p=0.038).

Conclusion: Increased adrenergic signaling promotes cervical cancer progression. Disruption of this pathway could provide a novel complement to current therapies.

Citation Format: Nouara C. Sadaoui, Guillermo N. Armaiz-Pena, Archana S. Nagaraja, Rajesha Rupaimoole, Rebecca A. Previs, Heather J. Dalton, Mangala S. Lingegowda, Lois M. Ramondetta, Anil K. Sood, Susan K. Lutgendorf, Steve W. Cole. Sustained adrenergic signaling promotes cervical cancer progression. [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 3511. doi:10.1158/1538-7445.AM2014-3511
Abstract 3520: TAZ (WWTR1), a key transcription co-activator of hippo-pathway, promotes hepatocellular carcinoma progression via PI3K/Akt/mTOR pathway
Authors: Hayashi Hiromitsu , Kuroki Hideyuki , Nakagawa Shigeki , Higashi Takaaki , Sakamoto Keita , Yokoyama Naomi , Ishiko Takatoshi , Beppu Toru , Baba Hideo
Published: May 2015

Introduction: The contact inhibition of proliferation is crucial for well-controlled organogenesis. Its disruption results in sustained cell proliferation, which is a hallmark of solid tumors. The Hippo signaling pathway has been implicated in contact inhibition of proliferation. Here, we investigated the role of TAZ (WWTR1), a key transcription co-activator of hippo-pathway, in HCC progression. Methods and Results: In HCC patients, the high TAZmRNA expression examined by real time PCR showed worsen survival outcomes compared with low expression group. Additionally, the tumor size in High TAZmRNA expression group was significantly larger than that in low expression group, and its high protein expression in HCC was deeply associated with the cell proliferative activity. A knockdown of TAZ expression in HCC cell lines attenuated the cell growth accompanying the downstream gene expressions (CTGF and CYR61). As the TAZ-mediated signaling pathway in cell growth, a knockdown of TAZ expression using RNAi inactivated the PI3K/Akt/mTOR pathway. Conclusion: Thus, TAZ plays a crucial role in tumor progression by accelerating cell growth via a PI3K/Akt/mTOR pathway, and tumoral TAZmRNA expression level may be a useful prognostic biomarker in HCC.

Citation Format: Hiromitsu Hayashi, Hideyuki Kuroki, Shigeki Nakagawa, Takaaki Higashi, Keita Sakamoto, Naomi Yokoyama, Takatoshi Ishiko, Toru Beppu, Hideo Baba. TAZ (WWTR1), a key transcription co-activator of hippo-pathway, promotes hepatocellular carcinoma progression via PI3K/Akt/mTOR pathway. [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 3520. doi:10.1158/1538-7445.AM2014-3520
Abstract 3527: YYEI motif is critical to oncogenicity of 14-3-3 proteins
Authors: Chang Wen-Hsin , Chen Ching-Hsien , Hong Qi-Sheng , Chen Jian-Wei , Yu Sung-Liang , Chen Chiung-Tong
Published: May 2015

14-3-3 family consists of seven highly conserved isoforms and most of them are identified as oncogenes in various types of cancer except for 14-3-3σ, a well-known tumor suppressor. To clarify the suppressor characteristic of 14-3-3σ divergent from others, we found that the major difference between the other oncogenic members and 14-3-3σ by protein alignment was an amino acid substitution (Y180H) by which the SH2-binding motif (YYEI) is disrupted and cannot be phosphorylation. Thus, we generated a H180Y 14-3-3σ mutant and investigated the impact of the acquired YYEI motif on the tumor suppression. First, we found that 14-3-3σ decreased cancer invasion. Surprisingly, the H180Y mutant not only enhanced cell invasion but increased cell viability. Meanwhile, the interaction between H180Y mutant and Src was higher than wild type. It indicates one amino acid substitution switches 14-3-3σ from tumor suppressor to oncogene. Hence, the YYEI motif might be important for the oncogenicity of other 14-3-3 proteins. Indeed, we demonstrated that 14-3-3ζ interacted with Src through Y178 phosphorylation, which is crucial for the binding of 14-3-3ζ with Src-SH2 domain. Owing to the importance of Y178 phosphorylation in the 14-3-3ζ/Src interaction, we introduced the Y178F 14-3-3ζ mutant to lung cancer cells and confirmed that Y178 phosphorylation is important for the increase of invasion and viability. Furthermore, we also revealed an amino acid switch of 14-3-3σ from YYEI to HYEI during evolutionary progression. Taken together, our findings suggest that YYEI motif is essential for 14-3-3 proteins to interact with Src and to regulate Src-mediated cell functions.

Citation Format: Wen-Hsin Chang, Ching-Hsien Chen, Qi-Sheng Hong, Jian-Wei Chen, Sung-Liang Yu, Chiung-Tong Chen. YYEI motif is critical to oncogenicity of 14-3-3 proteins. [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 3527. doi:10.1158/1538-7445.AM2014-3527
Abstract 3533: ADAM9 regulates microRNAs expression for lung cancer metastasis
Authors: Sher Yuh-Pyng , Wang Li-Ju , Tsai Mong-Hsun , Kuo Ting-Ting , Chuang Eric Y. , Lai Liang-Chuan
Published: May 2015

Lung cancer is the leading cause of cancer death worldwide, and brain metastasis is a major cause of morbidity and mortality in lung cancer. Currently, it is not clear which molecules or what mechanisms mediate lung cancer brain metastasis. To explore this question, we compared our established brain-metastatic lung cancer sublines with their parental cancer cells, and identified ADAM9 (a disintegrin and metalloprotease 9) was overexpressed in metastatic sublines. Knocking down ADAM9 expression in lung cancer cells also showed significant reduction of lung cancer brain metastasis in vitro and in vivo. Furthermore, using a genome-wide approach to screen ADAM9-related mRNAs and microRNAs involved in brain metastasis, we identified that CDH2, encoding N-cadherin, was up-regulated by ADAM9 in brain-metastatic lung cancer sublines, and the 3′UTR of CDH2 contains the predicted binding sites of several microRNAs that were down-regulated in ADAM9-overexpressed cancer cells. Luciferase assays and western blot analysis showed that CDH2 is a target gene of microRNAs. The up-regulation of microRNAs, in turn, reduced the expression of its target gene and further inhibited the migration ability of aggressive lung adenocarcinoma cells. This study revealed that ADAM9 activates CDH2 through the release of microRNA inhibition of CDH2 in lung adenocarcinoma and plays a role in lung cancer metastasis.

Citation Format: Yuh-Pyng Sher, Li-Ju Wang, Mong-Hsun Tsai, Ting-Ting Kuo, Eric Y. Chuang, Liang-Chuan Lai. ADAM9 regulates microRNAs expression for lung cancer metastasis. [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 3533. doi:10.1158/1538-7445.AM2014-3533
Abstract 3542: Upregulation of microRNA-372 & 373 associates with cervical lymph node metastasis and poor prognosis of oral carcinomas
Authors: Liu Chung J. , Tu Hsi-Feng , Cheng Hui-Wen , Chang Kuo-Wei
Published: May 2015

Purpose: Oral squamous cell carcinoma (OSCC) is prevalent worldwide, and survival in OSCC has not improved significantly in the last few decades. MicroRNAs (miRNAs) have an important regulatory role in human cancer including oral carcinogenesis. MicroRNA 372 and microRNA 373 play as oncogenic and tumour-suppressive functions of between different human maligngncy. This study investigated the microRNA-372 and microRNA 373 expression and their clinical implication in OSCC.

Experimental Design: Fifty patients with primary OSCC were included in the study. Primary cancer cells and matched normal oral epithelium were purified by laser capture microdissection. RNA were extracted from these samples. The expression levels of miR-372 and miR-373 in the tissue of OSCC patients were measured by quantitative RT-PCR.

Results: both miR-372 and miR-373 was up-regulated in OSCC tissue relative to control mucosa. Among different clinical variables, over-expression of miR-372 and miR-373 were associated with nodal metastasis, lymphovascular invasion and poor survival. Multivariate analysis showed that both high miR-372 and miR-373 expression were independent predictors for poor survival in OSCC.

Conclusion: Over expression of miR-372 and miR-373indicate worse survival in OSCC.

Citation Format: Chung Ji Liu, Hsi-Feng Tu, Hui-Wen Cheng, Kuo-Wei Chang. Upregulation of microRNA-372 & 373 associates with cervical lymph node metastasis and poor prognosis of oral carcinomas. [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 3542. doi:10.1158/1538-7445.AM2014-3542
Abstract 3543: A long noncoding RNA plays a critical role in Bcr-Abl-mediated cellular transformation
Authors: Guo Guijie , Kang Qingzheng , Chen Qinghuang , Jing Ouyang , Chen Jilong
Published: May 2015

It is well documented that malignant transformation of myeloid and lymphoid cells by Bcr-Abl involves the dysregulation or mutation of a variety of protein-coding genes that are normally involved in regulating the proliferation and survival of hematopoietic cells. However, the functional relevance of long noncoding RNAs (lncRNAs) and their in vivo regulation during Bcr-Abl-mediated tumorigenesis remain unknown, although recent studies have shown that aberrant expression of lncRNAs is associated with various human cancers. In this study, we performed a comprehensive analysis of lncRNAs in human CML cells using an lncRNA cDNA microarray, and identified an lncRNA termed lncRNA-BGL3 that acted as a key regulator of Bcr-Abl-mediated cellular transformation. Notably, we observed that lncRNA-BGL3 was highly induced in response to disruption of Bcr-Abl expression or by inhibiting Bcr-Abl kinase activity in K562 cells and leukemic cells derived from CML patients. Ectopic expression of lncRNA-BGL3 sensitized leukemic cells to undergo apoptosis and inhibited Bcr-Abl-induced tumorigenesis. Furthermore, transgenic mice expressing lncRNA-BGL3 were generated. We found that transgenic expression of lncRNA-BGL3 alone in mice was sufficient to impair primary bone marrow transformation by Bcr-Abl. Interestingly, we identified that lncRNA-BGL3 was a target of miR-17, miR-93, miR-20a, miR-20b, miR-106a and miR-106b, microRNAs that repress mRNA of PTEN. Further experiments demonstrated that lncRNA-BGL3 functioned as a competitive endogenous RNA (ceRNA) for binding these microRNAs to cross-regulate PTEN expression. Additionally, our experiments have begun to address the mechanism of how lncRNA-BGL3 is regulated in the leukemic cells and showed that Bcr-Abl repressed lncRNA-BGL3 expression through c-Myc-dependent DNA methylation. Taken together, these results reveal that Bcr-Abl-mediated cellular transformation critically requires silence of tumor suppressor lncRNA-BGL3 and suggest a potential strategy for the treatment of Bcr-Abl-positive leukemia.

Citation Format: Guijie Guo, Qingzheng Kang, Qinghuang Chen, Ouyang Jing, Jilong Chen. A long noncoding RNA plays a critical role in Bcr-Abl-mediated cellular transformation. [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 3543. doi:10.1158/1538-7445.AM2014-3543
Abstract 3544: Tumor suppressor function of loc285194 in breast cancer
Authors: CHI YAYUN , Huang Sheng , Wu Jiong
Published: May 2015

Accumulating studies have focused on the oncogenic and tumor suppressive roles of the newly identified lncRNAs. LOC285194 was found to be associated with poor survival of osteosarcoma patients and colorectal cancer patients. Also loc285194 is reported to be a direct transcription target of p53 and could suppress cell growth. However, its role in breast cancer (BC) remains unknown. The aim of this study was to examine the expression pattern of loc285194 in BC and to evaluate its biological role and clinical significance in prediction of prognosis. In this study, we examined expression of loc285194 in 20 pairs of BC cancer tissues and adjacent noncancerous tissues (ANCT), also in 151 BC tissues, 14 BC cell lines and one normal breast cell line. It showed that loc285194 expression was lower both in BC samples and in BC cell lines compared with corresponding normal control. Loc285194 expression was correlated with menopausal status. Postmenopausal patients had a lower loc285194 expression level comparing with premenopausal patients (p=0.040). Moreover, low loc285194 expression was correlated with a relatively poor survival status (p=0.023) and a poor tumor differentiation (p=0.035). The Kaplan-Meier survival curves indicated that the overall survival (OS) was significantly poor in BC patients who showed low loc285194 expression. Furthermore, expression of loc285194 is significantly associated with worse prognosis independent of prognostic markers in estrogen receptor (ER) negative tumor samples (p = 0.05). Our studies indicate that loc285194 may represent a new marker of prognosis and it is a potential therapeutic target for breast cancer especially for ER negative breast cancer.

Citation Format: YAYUN CHI, Sheng Huang, Jiong Wu. Tumor suppressor function of loc285194 in breast 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 3544. doi:10.1158/1538-7445.AM2014-3544
Abstract 3559: Long non-coding RNA subtype classification of clear-cell renal cell carcinoma
Authors: Malouf Gabriel G. , Zhang Jianping , Tannir Nizar , Thompson Erika J. , Khayat David , Spano Jean-Philippe , Su Xiaoping
Published: May 2015

Background: Long non-coding RNAs (lncRNA) play a key role in regulating cell physiology through different mechanisms including regulation of transcription and splicing, and recruitment of histone-modifying enzymes. The portrait of lncRNA in kidney cancers remains fully unknown.

Material and Methods: we performed a genomic analysis of GENCODE lncRNAs in clear-cell renal cell carcinoma (ccRCC), using The Cancer Genome Atlas (TCGA) molecular RNAseq profiles of 475 primary tumors. Furthermore, we described global correlations between lncRNA and expression of cis- and trans-acting genes, and established lncRNA expression subtypes based on distinct signatures. In addition, lncRNA expression was correlated with miRNA, copy-number alterations and the chromatin landscape of human kidney.

Results: Using stringent criteria (RPKM ≥1 in at least 10% of ccRCC), we identified 1,934 lncRNAs and predicted those that are potential drivers of cancer progression through integrative analysis. The expression of 1,057 (54.6%) lncRNAs showed a positive correlation with the mRNA expression of their neighboring genes, while only 13 (0.7%) lncRNAs showed a negative correlation. Overall, 844 (43.6%) lncRNAs were pre-marked by H3K4me3 in normal kidney, suggesting that those lncRNA might arise from promoter-associated elements. Conversely, only 81 (4.2%) and 182 (9.4%) lncRNAs were pre-marked by H3K36me3 and H3K27me3, respectively. Unsupervised clustering of lncRNA expression revealed four robust subtypes, which differ from the ccRCC transcriptomic subgroups. Interestingly, patients belonging to cluster II (n=111) displayed poor overall survival as compared to patients of other clusters (p=2.6x10-6). Intriguingly, there were 37 (7.8%) ccRCC belonging to cluster IV, and those were enriched for TFE3/TFEB RCC. Furthermore, tumors within cluster IV were devoid from 3p loss and 5q gain which represent the most frequent genomic aberrations in ccRCC. Finally, GREAT analysis reveals that overexpressed lncRNA in cluster IV were exclusively enriched for genes belonging to collecting duct (p=1.6x10-13) highly suggesting that their cell of origin might be different.

Conclusion: This study describes the first portrait of lncRNA in ccRCC and proposes a robust lncRNA subtype classification of ccRCC, which may serve in predicting patient outcome. Furthermore, we identified a small ccRCC subgroup with distinct lncRNA and karyotypic phenotype.

Citation Format: Gabriel G. Malouf, Jianping Zhang, Nizar Tannir, Erika J. Thompson, David Khayat, Jean-Philippe Spano, Xiaoping Su. Long non-coding RNA subtype classification of clear-cell renal cell carcinoma. [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 3559. doi:10.1158/1538-7445.AM2014-3559
Abstract 3561: Whole transcriptome analysis of testicular germ cell tumors
Authors: Degoricija Lovorka , Lee Kathy Y. , Patel Sunali , Chu Shirley , Gillis Ad J.M. , Rijlaarsdam Martin , Dorssers Lambert C.J. , Looijenga Leendert
Published: May 2015

Next generation sequencing of the whole transcriptome enables high resolution measurement of gene expression activity in different tissue and cell types. This methodology provides an in depth study of known transcripts and depending on the data analysis, allows identification of additional transcript types such as transcript variants, fusion transcripts, and small and long ncRNAs. In this study we performed RNA-Seq using the Ion Torrent sequencing platform to compare the expression profile of testicular germ cell tumors (seminoma type, n=3) and normal testis (n=3). Using Partek Flow and Star or TopHat aligners, we aligned the reads to the human genome and mapped sequences to the RefSeq database. We identified a large number of genes that were up and down regulated with high degree of significance p<0.01, >2X FC). These included genes related to testicular tissue type, stem cell pleuripotency (NANOG; POU5F1) and proliferation (KRAS, CCND2). In addition, a number of differentially expressed noncoding RNAs were identified (SNORD12B, XIST). The method was validated on a small set of genes (>20) using qPCR (TaqMan Assays). We used the Open Array platform to quantitatively screen a larger number of differentially expressed genes (224) across a number of different testicular germ cell tumor types (non-seminoma).

Citation Format: Lovorka Degoricija, Kathy Y. Lee, Sunali Patel, Shirley Chu, Ad J.M. Gillis, Martin Rijlaarsdam, Lambert C.J. Dorssers, Leendert Looijenga. Whole transcriptome analysis of testicular germ cell 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 3561. doi:10.1158/1538-7445.AM2014-3561
Abstract 3574: Analysis of whole genome and transcriptome sequencing in single cell
Authors: Kwon Nak-Jung , Lee Woo Chung , Kim Jiwoong , Kim Hyeri , Seong Ahreum , Kim Bong Cho , Park Doo Hyun , Yang Kap-Seok
Published: May 2015

The rapid advancement in Next Generation Sequencing (NGS) technology has allowed extensive genetic profiling and analysis of tumor tissues. While accurate tumor characterization is crucial for cancer therapy, current sequencing methods are unable to resolve the genetic differences between the heterogeneous cell populations within tumor. By profiling specific subpopulations of cells from a larger heterogeneous population, single cell sequencing can improve the efficiency of cancer treatments through early detection of rare tumor cells, monitoring of circulating tumor cells, and accurate profiling of intra-tumor heterogeneity. In this study, we compared commercially available single cell amplification methods and sequencing platforms for whole genome and whole transcriptome sequencing. We isolated single cells from a human breast cancer cell line, MCF7, and human embryonic kidney cell line, HEK293, by micromanipulation, and their whole genome and transcriptome were sequenced. For whole genome sequencing, the performance of Repli-g (Qiagen), Ampli1 (Slicon Biosystems) and WGA4 (Sigma) were assessed for whole genome amplification. Next, we used TruSeq DNA Library Kit and Ion Plus Fragment Library Kit for library preparation, and whole genome sequencing was performed on HiSeq2000 and Proton. The sequencing data were analyzed by SAMtools/GATK and tmap-f3. For whole transcriptome sequencing, cDNA was synthesized from single cells using SMARTer ultra low input RNA kit (Clontech), and the cDNA library was prepared with Nextera XT library prep kit (Illumina) and Ion Total RNA-Seq Kit v2 (Life Technologies). Sequencing was performed on HiSeq2000 and Proton, and analyzed with Tophat and Cufflink. We show that the percentage of mappable reads and regions with more than 1X coverage in whole genome sequencing were similar to that of the genomic DNA data, but the coverage rate of regions with more than 10X was lower in single cell data on both NGS platforms. In the RNA-Seq, the gene detection rate in single cell and more than 100 cells were about 65% and 85% compare to total RNA, respectively.

Citation Format: Nak-Jung Kwon, Woo Chung Lee, Jiwoong Kim, Hyeri Kim, Ahreum Seong, Bong Cho Kim, Doo Hyun Park, Kap-Seok Yang. Analysis of whole genome and transcriptome sequencing in single cell. [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 3574. doi:10.1158/1538-7445.AM2014-3574
Abstract 3580: Hotspot mutation and fusion transcript detection from the same non-small lung adenocarcinoma sample
Authors: Cheng Angie , Bagai Varun , Cienfuegos Joey , Hernandez Natalie , Li Mu , Schageman Jeff , Fekete Richard , Petraroli Rosella , Vlassov Alexander , Magdaleno Susan
Published: May 2015

The presence of certain chromosomal rearrangements and the subsequent fusion gene derived from translocations has been implicated in a number of cancers. Hundreds of translocations have been described in the literature recently but the need to efficiently detect and further characterize these chromosomal translocations is growing exponentially. The two main methods to identify and monitor translocations, fluorescent in situ hybridization (FISH) and comparative genomic hybridization (CGH) are challenging, labor intensive, the information obtained is limited, and sensitivity is rather low. Common sample types for these analyses are biopsies or small tumors, which are very limited in material making the downstream measurement of more than one analyte rather difficult; obtaining another biopsy, using a different section or splitting the sample can raise issues of tumor heterogeneity. The ability to study mutation status as well as measuring fusion transcript expression from the same sample is powerful because you're maximizing the information obtained from a single precious sample and eliminating any sample to sample variation. Here we describe the efficient isolation of two valuable analytes, RNA and DNA, from the same starting sample without splitting, followed by versatile and informative downstream analysis. This methodology was applied to FFPE and degraded samples as well as fresh tissues and cells. DNA and RNA were recovered from the same non-small lung adenocarcinoma sample and both mutation analysis, as well as fusion transcript detection was performed using the Ion Torrent PGM™ platform on the same Ion 318™ chip. Using 10ng of DNA and 10ng of RNA input, we applied the Ion AmpliSeq™ Colon and Lung Cancer panel to analyze over 500 COSMIC mutations in 22 genes and the Ion AmpliSeq™ RNA Lung Fusion panel to detect 40 different fusion transcripts.

Citation Format: Angie Cheng, Varun Bagai, Joey Cienfuegos, Natalie Hernandez, Mu Li, Jeff Schageman, Richard Fekete, Rosella Petraroli, Alexander Vlassov, Susan Magdaleno. Hotspot mutation and fusion transcript detection from the same non-small lung adenocarcinoma sample. [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 3580. doi:10.1158/1538-7445.AM2014-3580
Abstract 3582: A simple multiplex PCR approach for target enrichment in next-gen sequencing
Authors: Zhou Xiaochuan , Zhu Qi , Hebel Chris
Published: May 2015

Multiplexing PCR is a simple way to extract genomic regions of interest for various medical and genetic tests. Somatic mutations lead to various diseases including cancer. These mutations are unlikely to be best detected using regular whole genome sequencing. Clinical samples often consist of disease cells, e.g. cancer cells, surrounded by normal cells. Thus, deep sequencing of hundreds to thousands fold coverage is required to detect the mutations. In clinical research many doctors are interested in specific genes or genomic regions and they want to extract the regions from genomic DNA or RNA before sequencing. Many current clinical, forensic, and heretical genetic test workflows start with multiplexing PCR to extract genetic marker carrying regions from whole genomes before running hybridization, sequencing, or electrophoresis tests to identify the markers. Personal medicine and prognosis mostly involve examining sequence variations of a number of targeted genes and metabolic pathway genes so as to predict drug efficacies and drug toxicities.

We have developed a new multiplexing PCR approach with a significantly simplified workflow and significantly improved robustness. When applied to sequencing target enrichment application, the workflow for producing amplified targets involves only one hands-on step and one PCR run. The approach is designed to require low sample input and to produce superior amplicon uniformity and sequence specificity. The approach involves a novel primer design and a proprietary reaction composition. A PCR run consists of two functionally separated reaction phases, namely target capture and library amplification, without any hands-on step in between. The performance of the new approach will be demonstrated by a caner panel data.

Citation Format: Xiaochuan Zhou, Qi Zhu, Chris Hebel. A simple multiplex PCR approach for target enrichment in next-gen sequencing. [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 3582. doi:10.1158/1538-7445.AM2014-3582
Abstract 3584: Analysis of colorectal intratumoral genetic heterogeneity by high efficiency and rapid deep targeted sequencing
Authors: Hopmans Erik S. , Lee Hojoon , Miotke Laura , Rumma Rowza Tur , Grimes Sue , Bell John M. , Ji Hanlee P.
Published: May 2015

Metastasis is the most frequent cause of morbidity and mortality in cancer. Metastatic disease lethality is often the result of cancer progression despite treatment. Therapeutic resistance is seen nearly in all cases of metastatic cancer. Genetic studies relying on deep sequencing have shown that many cancers are genetically heterogeneous as a result of diverse clonal populations with delineating and unique set of mutations. Several studies suggest that resistance mutations are present in tumors before the start of treatment and via evolution, clonal populations expand under therapeutic selection. However, these minor allelic mutations that define clonal populations are not typically detectable with typical sequencing coverage (20-50X) employed in whole genome approaches.

Relying on an innovative programmable targeting method that enables us to rapidly configure nearly any region of the human genome and efficiently sequence cancer genomes, we are determining the extent of intratumoral genetic heterogeneity and clonal diversity in colorectal cancer. Our study involves over 120 patients with available clinical data and a subset of matched tumors indicative of tumor progression (e.g. tumor-normal, premalignant-malignant, malignant-metastasis). Via an integrative analysis of TCGA, COSMIC and other genomic data sets of colorectal cancer, we identified the top ranking 53 cancer genes prone to mutation and associated with advanced colorectal cancer by. These top genes are found in known cancer pathways such as the WNT and RAS/RAF pathway and we are analyzing the exons of these genes for mutations, insertions and deletions with a sequencing depth of at least 1000x. Preliminary results have shown that we can readily reach sequencing depths of 7000x with our automated targeting approach and that a minimal sequencing depth of 1000x will enable detection of aberrations present in the sample below 1%. Samples are being analyzed for the overall level of heterogeneity and correlation of clonal mutations to clinical outcome.

For optimal personalized treatment of cancer patients, the analysis of intratumoral genetic heterogeneity may be useful in predicting treatment response and metastatic potential of any given primary colorectal cancer.

Citation Format: Erik S. Hopmans, Hojoon Lee, Laura Miotke, Rowza Tur Rumma, Sue Grimes, John M. Bell, Hanlee P. Ji. Analysis of colorectal intratumoral genetic heterogeneity by high efficiency and rapid deep targeted sequencing. [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 3584. doi:10.1158/1538-7445.AM2014-3584
Abstract 3591: 15-Deoxy-Δ12,14-prostaglandin J2-induced epithelial-to-mesenchymal transition in human breast cancer cells promotes fibroblast activation
Authors: Kim Do-Hee , Choi Jeehye , Suh Jin-Young , Na Hye-Kyung , Surh Young-Joon
Published: May 2015

In inflammation-associated carcinogenesis, cyclooxygenase-2 (COX-2) is markedly overexpressed, resulting in accumulation of various prostaglandins. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and prostahlandin E2 (PGE2) are the predominant COX-2-derived prostaglandins (PGs) with oncogenic potential. In the present study, we examined the effects of these to PGs on epithelial-to-mesenchymal transition (EMT), a process by which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties. 15d-PGJ2 induced EMT to a greater than PGE2 in human breast cancer (MCF-7) cells. Snail and ZEB1 are known to bind to the E-boxes present in the E-cadherin promoter, thereby repressing E-cadherin transcription. Treatment of MCF-7 cells with 30 μM of 15d-PGJ2 increased the expression of Snail and ZEB1, followed by the down-regulation of E-cadherin. Nuclear extract from 15d-PGJ2-treated MCF-7 cells showed the binding of Snail and ZEB1 to E-box sequences in the E-cadherin promoter. In addition, among various cytokines/chemokines examined, the mRNA level of interleukin-8 (CXCL8) was highly elevated in 15d-PGJ2-stimulated MCF-7 cells. 15d-PGJ2-induced up-regulation of CXCL8 expression was abrogated by the introduction of Snail short interfering RNA. These findings suggest that CXCL8 may function as a positive regulator of EMT in 15d-PGJ2-treated MCF-7 cells. Moreover, the addition of conditioned medium of MCF-7 cells undergoing EMT activates normal fibroblast cells to cancer-associated fibroblasts (CAFs). Co-culture of normal fibroblasts with 15d-PGJ2-treaed-MCF-7 cells also induced the expression of α-SMA and FAPα, representative markers of CAFs. As a corresponding receptor of CXCL8, CXCR2 was overexpressed in activated fibroblasts co-cultured with 15d-PGJ2-treated MCF-7 cells. Unlike 15d-PGJ2, its non-electrophilic analogue 9,10-dihydro-15d-PGJ2 failed to induce EMT, suggesting that the α,β-unsaturated carbonyl group located in the cyclopentenone ring of 15d-PGJ2 is essential for EMT induction. Notably, activation of Rho kinase through direct binding of ArhGEF5, a guanine nucleotide exchange factor, with 15d-PGJ2 seems to contribute to the EMT induction. Taken together, above findings suggest that 15d-PGJ2 induces EMT through up-regulation of Snail expression and subsequent production of CXCL8 which may contribute to increased EMT of human breast cancer cells.

Citation Format: Do-Hee Kim, Jeehye Choi, Jin-Young Suh, Hye-Kyung Na, Young-Joon Surh. 15-Deoxy-Δ12,14-prostaglandin J2-induced epithelial-to-mesenchymal transition in human breast cancer cells promotes fibroblast activation. [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 3591. doi:10.1158/1538-7445.AM2014-3591
Abstract 3593: Examining the role of tumor cell secreted factors in intravasation and enhancing paracrine loop invasion
Authors: Cabrera Ramon M. , Zhou Zhen N. , Roh-Johnson Minna , Condeelis John , Cox Dianne , Segall Jeffrey E.
Published: May 2015

The interaction of breast cancer cells with other cells in the tumor microenvironment plays an important role in metastasis. Invasion and intravasation, two critical steps in the metastatic process, are thought to be influenced by these interactions in addition to their intrinsic invasive properties. Macrophages are of particular interest when it comes to studying tumor cell invasiveness. Previous studies have shown that there is paracrine loop signaling between breast cancer cells and macrophages in which colony stimulating factor 1 (CSF-1) produced by tumor cells stimulates epidermal growth factor (EGF) production by macrophages. This EGF, in turn, stimulates invasion of the tumor cells. In addition to the classically described paracrine loop, it has been seen that tumor cell expression of ErbB3, a member of the EGFR family of receptor tyrosine kinases, may play a role in facilitating tumor cell intravasation. Overexpression of ErbB3 in breast cancer patients correlates with decreased survival. We show that using a blocking antibody against ErbB3 results in a significant decrease in macrophage-induced transendothelial migration of breast cancer cells. Tumor cell secretion of Neuregulin 1 (NRG1), a ligand of the ErbB3 receptor, may also play a role in the tumor cell- macrophage paracrine interaction. We show that stimulation of macrophages with NRG1 upregulates mRNA expression of JAG1, a ligand of the Notch receptor. Activation of the Notch receptor pathway has been shown to be involved in the process of tumor cell invasion. Overall, our studies look to examine this interaction between tumor cells and macrophages and these observations indicate that ErbB3, NRG1, and JAG1 could serve as novel targets in metastasis and the tumor microenvironment.

Citation Format: Ramon M. Cabrera, Zhen N. Zhou, Minna Roh-Johnson, John Condeelis, Dianne Cox, Jeffrey E. Segall. Examining the role of tumor cell secreted factors in intravasation and enhancing paracrine loop invasion. [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 3593. doi:10.1158/1538-7445.AM2014-3593
Abstract 3594: Tumor microenvironment derived exosomes regulate prostate cancer metabolism
Authors: Zhao Hongyun , Yang Lifeng , Achreja Abhinav , Marini Juan , Peehl Donna , Nagrath Deepak
Published: May 2015

Tumor microenvironment is composed of myofibroblasts, extracellular matrix, and many other cells. There are many ways for tumor to communicate with its microenvironment, such as growth factors, cytokines, chemokines and so on. Recent research has indicated that exosomes may serve an important role in communication between tumor and its microenvironment. Exosomes are nanosized particles secreted by cells and contain microRNA, metabolic enzymes, biomarker proteins, and some other functional components. We show a novel regulatory mechanism for modulating metabolism in prostate cancer cells by stromal cells. We found that exosomes from stromal fibroblast cells are uptaken by prostate cancer cells and they release RNAs, enzymes to regulate cancer cells metabolism. Our result shows that exosomes from patient derived fibroblasts enhance glucose uptake, lactate production and pyruvate uptake in prostate cancer cells. We further performed 13C based isotope labeling experiments using glucose and glutamine labeling to study metabolic rewiring induced in cancer cells by stromal exosomes. We found that exosomes downregulated mitochondria metabolism in prostate cancer cells. In summary, stroma derived exosomes enhanced Warburg effect in prostate cancer cells. Our findings can lead to the development of new therapeutics integrating tumor microenvironment and metabolic pathways in tumor cells.

Citation Format: Hongyun Zhao, Lifeng Yang, Abhinav Achreja, Juan Marini, Donna Peehl, Deepak Nagrath. Tumor microenvironment derived exosomes regulate prostate cancer metabolism. [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 3594. doi:10.1158/1538-7445.AM2014-3594
Abstract 3596: The role of PIM1 kinase in the prostate tumor microenvironment
Authors: Zemskova Marina , Kraft Andrew S.
Published: May 2015

Prostate cancer (PCa) remains the second leading cause of cancer-related deaths in men. The prostate is a tubulo-alveolar gland composed of epithelial tissues embedded in stromal components. PCa includes reactive stroma which consists of myofibroblasts and extracellular matrix (ECM) remodeling. Increasing evidence suggests that myofibroblasts may refine the prognostic assessment of tumors. The PIM1 serine/threonine protein kinase is a true oncogene and has been implicated in the initiation and progression of multiple cancer types including PCa. The level of PIM1 correlates with the disease state and Gleason grade and has been shown to stimulate growth of murine prostate cancer. The role of PIM1 in prostate stromal cells has never been investigated. Using human prostate tissue microarray analysis we now demonstrate that (1) the PIM1 protein is detected in human stroma adjacent to cancer tissue; (2) stromal cells isolated from PIM1 knockout mice contain a decreased number of myofibroblasts and expose slow growth rate when compared with wild type control, suggesting that kinase expression can alter stromal cell composition and proliferation; (3) elevated level of PIM1 kinase in human benign prostate stromal cells increases migration and growth of prostate epithelial cells and this effect can be reversed by PIM inhibitors; (4) PIM1 over-expressing stromal cells increase epithelial cell growth in tissue regeneration model; (5) stromal cells with elevated level of PIM1 expose increased number of myofibroblasts when co-cultivated with prostate epithelial cells. These data suggest that PIM1 kinase may contribute in formation of a “reactive stroma” phenotype. We used the conditioning media analysis to decipher the PIM1 specific signal transduction pathways that may lead to stroma activation and drive prostate cancer growth. Using cytokine/chemokine arrays and ECM protein analysis we found that overexpression of PIM1 kinase in stromal cells is associated with elevated level of CCL5 chemokine and increased secretion of collagen1. Examination of the pathways involved in formation of myofibroblasts revealed that PIM1 can modulate activation of PDGFRβ leading to the increased CCL5 expression. We are suggesting that PIM1 is a modulator of stromal cells response to the stimuli produced by prostate epithelial cells through increase of matrix stiffness, receptor activation and accumulation of myofibroblasts which induce further ECM remodeling and expression of the growth factors leading to epithelial cells transformation and cancer propagation.

Citation Format: Marina Zemskova, Andrew S. Kraft. The role of PIM1 kinase in the prostate tumor microenvironment. [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 3596. doi:10.1158/1538-7445.AM2014-3596
Abstract 3597: Malignancy of bladder cancer cells is enhanced by tumor associated fibroblasts through a cytokine-chemokine loop
Authors: Grimm Susanne , Jennek Susanne , Bratsch Jens , Wohlmann Andreas , Mohta Vrinda , Junker Kerstin , Friedrich Karlheinz
Published: May 2015

The tumor microenvironment plays an important role in cancer progression. In particular, stromal fibroblasts have profound influence on migration and invasiveness of tumor cells. We employ bladder carcinoma cell lines of different de-differentiation grade to pursue the hypothesis that tumor cell malignancy correlates with the cells' potential to recruit support by tumor associated fibroblasts (TAFs).

Bladder cancer cell lines RT112 and Cal29 were used as models for tumor cells of low or high de-differentiation grade, respectively. They were grown separately or in co-culture with tumor-associated fibroblast isolated from kidney carcinomas. Migratory and invasive cell properties were quantified by Boyden chamber (Matrigel) and wound healing assays. Cytokine production of cells was measured by intracellular staining followed by cytometry, by semi-quantitative PCR, or by subjecting conditioned medium to ELISA and Western blot analysis.

Migration and invasiveness of bladder cancer cell lines Cal29 and RT112 was enhanced by co-cultivation with TAFs. The low grade cell line RT112, however, was much more dependent on TAF-derived signals to increase the malignancy parameters. Conditioned medium from tumor cells induced the secretion of Matrix Metalloproteinases (MMPs) and the release of GM-CSF, HGF, and Monocyte Chemotactic Protein (MCP-1, CCL2) by TAFs. We could identify tumor cell-derived IL-1α and β as well as TGF-β as stimuli responsible for the upregulation of GM-CSF and MCP-1, respectively, in fibroblasts. MCP-1 is implicated in inflammatory disease states characterized by monocytic infiltrates and was shown to promote cell migration of both bladder cancer cell lines under investigation.

Employing pathway-specific reporter gene constructs we are currently addressing signal transduction reactions in both tumor cells and fibroblasts underlying this crosstalk.

Supported by: IZKF Jena, European Regional Development Fund ERDF.

Citation Format: Susanne Grimm, Susanne Jennek, Jens Bratsch, Andreas Wohlmann, Vrinda Mohta, Kerstin Junker, Karlheinz Friedrich. Malignancy of bladder cancer cells is enhanced by tumor associated fibroblasts through a cytokine-chemokine loop. [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 3597. doi:10.1158/1538-7445.AM2014-3597
Abstract 3609: Fibroblast influence on keratinocyte tumorigenic and metastatic potential in cutaneous squamous cell carcinoma
Authors: Gwilliam J Curtis , CVitan Alexander , Watson Nicholas , Falkenberg Lauren , Yousefi Nyousha , Alexander Hepzibha , Morin Adam , Wiest Jonathan S. , DeCicco-Skinner Katie L.
Published: May 2015

Skin cancer is the most common form of cancer in the United States, with an estimated two million cases diagnosed annually. Tpl2 (Map3k8) is a serine threonine protein kinase in the mitogen-activated protein kinase (MAPK) signal transduction cascade. Tpl2 was identified by our laboratory as having a tumor suppressive function in skin carcinogenesis, with Tpl2 knockout mice developing significantly more tumors than matched wildtype controls and Tpl2 knockout keratinocytes having greater angiogenic and metastatic potential. However, the microenvironmental contributions of underlying fibroblasts to the cancerous behavior seen in Tpl2 knockout keratinocytes has yet to be characterized. Therefore, we compared the tumorigenic, invasive, and metastatic potential of H-ras infected keratinocytes from wildtype and Tpl2 knockout mice with H-ras infected keratinocytes co-cultured with fibroblasts from wildtype and Tpl2 knockout mice. Using various techniques, including protein microarray, real-time polymerase chain reaction and Western blotting we found that fibroblasts in the microenvironment of H-ras-infected Tpl2 knockout keratinocytes contribute to an up-regulation of markers involved in growth and progression of squamous cell carcinoma. Additionally, Tpl2 knockout keratinocytes cultured in fibroblast conditioned media (FCM) have higher malignant conversion rates, colony formation and increased angiogenesis compared to co-cultured wildtype keratinocytes. In summary, our studies demonstrate that fibroblasts in the tumor microenvironment of Tpl2 knockout mice play a major role in the more aggressive,invasive, and metastatic character of knockout tumors than is seen in control animals.

Citation Format: J Curtis Gwilliam, Alexander CVitan, Nicholas Watson, Lauren Falkenberg, Nyousha Yousefi, Hepzibha Alexander, Adam Morin, Jonathan S. Wiest, Katie L. DeCicco-Skinner. Fibroblast influence on keratinocyte tumorigenic and metastatic potential in cutaneous squamous cell carcinoma. [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 3609. doi:10.1158/1538-7445.AM2014-3609