bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2971: Methylation-mediated silencing of microRNAs during cervical carcinogenesis.
Authors: Wilting Saskia Marije , Verlaat Wina , Jaspers Annelieke , Makazaji Nour A. , Agami Reuven , Meijer Chris JLM , Snijders Peter JF , Steenbergen Renske DM
Published: August 2014

Deregulated expression of microRNAs (miRNAs) is common and biologically relevant in cervical carcinogenesis and appears only partly related to chromosomal changes. We recently identified 34 miRNAs showing decreased expression in cervical high-grade precursor lesions and carcinomas not associated with a chromosomal loss, 6 of which were located within a CpG island. This study aimed to investigate to what extent these miRNAs are subject to DNA methylation-mediated silencing in cervical carcinogenesis.

Methylation-specific PCR (MSP) analysis on a cell line panel representing different stages of human papillomavirus (HPV) induced transformation revealed an increase in methylation of hsa-miR-149, -203, and -375 with progression to malignancy, whereas expression of these miRNAs was restored upon treatment with a demethylating agent. All three miRNAs showed significantly increased levels of methylation in cervical carcinomas, whereas methylation levels of hsa-miR-203 and -375 were also significantly increased in high-grade cervical intraepithelial neoplasia (CIN). A pilot analysis showed that increased hsa-miR-203 methylation was also detectable in HPV-positive cervical scrapes of women with high-grade CIN compared to controls. Similar to recent findings on hsa-miR-375, ectopic expression of hsa-miR-203 in cervical cancer cells decreased both the proliferation rate and anchorage independent growth.

We found evidence for methylation-mediated silencing of hsa-miR-149, -203 and -375 in cervical cancer. Methylation of the latter two was already apparent in precancerous lesions and represent functionally relevant events in HPV-mediated transformation. In addition, as demonstrated for hsa-miR-203, these methylated miRNAs may provide valuable markers for assessment of the presence of (pre)cancerous cervical lesions in HPV-positive women.

Citation Format: Saskia Marije Wilting, Wina Verlaat, Annelieke Jaspers, Nour A. Makazaji, Reuven Agami, Chris JLM Meijer, Peter JF Snijders, Renske DM Steenbergen. Methylation-mediated silencing of microRNAs during cervical carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2971. doi:10.1158/1538-7445.AM2013-2971
Abstract 2975: Rad21, as a high order chromatin architecture molecule, regulates global gene transcription.
Authors: Yun Jiyeon , Song Sang-Hyun , Kang Jee-Youn , Kim Hwang-Phill , Han Sae-Won , Oh Do-Youn , Im Seock-Ah , Bang Yung-Jue , Kim Tae-You
Published: August 2014

Rad21 is a subunit of cohesin complex, which is well known to be involved in sister chromatid cohesion at mitotic phase. In recent studies, Rad21 has been also known to be associated with long-range chromosome interaction to dynamically regulate gene transcription. However, Very little is known about Rad21-mediated high order chromatin structure and global changes in gene transcription in human cancer.

In this study, we silenced Rad21 gene using Lenti-virus knockdown system and performed transcriptome analysis to verify the changes in gene transcription by knockdown of Rad21. Among genes showing significant transcription changes by the reduction of Rad21, we chose several target genes decreasing its transcription and checked the transcriptional change with passaging over a 60 day period. Interestingly, the change in transcription level of some genes appeared by the earliest passage time point, whereas those of other genes arose during the course of long-term passage. Consistent with gene transcription changes, results from a ChIP assay demonstrated that Rad21-knockdown cells showed low level of Rad21 occupancy and active histone marks (H3K4me3 and Pol2) on the target genes, indicating that dynamic histone modifications also contribute to regulation of the target genes.

To figure out whether the reduction of Rad21 binding directly affects the chromatin loops, we applied Chromosome Conformation Capture (3C) -qPCR in Rad21-knockdown cells. The results demonstrated that the decreased Rad21 expression cause overal reduction of locus interaction on the target genes, indicating that Rad21 binding was directly required for long-range interactions at the target gene regions and gene transcription regulation.

Taken together, the results of our study demonstrated that long-range chromosome interaction mediated by Rad21 can dynamically regulate global gene transcription in human cancer cells, indicating that it may be important for cancer development.

Citation Format: Jiyeon Yun, Sang-Hyun Song, Jee-Youn Kang, Hwang-Phill Kim, Sae-Won Han, Do-Youn Oh, Seock-Ah Im, Yung-Jue Bang, Tae-You Kim. Rad21, as a high order chromatin architecture molecule, regulates global gene transcription. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2975. doi:10.1158/1538-7445.AM2013-2975

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 298: Breast cancer malignancy and drug sensitivity are regulated by mammaglobin 1.
Authors: Picot Nadia , Beauregard Annie-Pier , Guerette Roxann , Jean Stéphanie , Robichaud Gilles A.
Published: August 2014

Breast cancer metastasis, a complex process enabling tumour cells dissemination, accounts for more than 90% of breast cancer mortality. The identification of genes involved in breast cancer metastasis is thus essential mitigate disease progression

One of these factors, mammaglobin 1 (MGB1), is specifically expressed in mammary epithelial tissues and overexpressed in mammary cancers. Normally, MGB1 plays an essential role in cellular homeostasis. More recently, studies show that MGB1 is a useful biomarker for prognostic application and the detection of metastastatic breast cancer. However, no studies reveal the cellular and molecular function of MGB1 in this disease. We therefore set out to elucidate the role of MGB1 in breast cancer processes.

We developed a breast cancer model with conditioned MGB1 expression to assess the molecular and cellular processes regulated by that protein which lead to breast cancer cell malignancy. After investigation, we observed that loss of MGB1 expression led to the reduction of cancer cell growth which was supported by the suppression of p38, JNK, ERK and NFκB activities. We also found that MGB1 promotes cell migration which is supported through increased activities of FAK and matrix metalloproteinases. MGB1 also seems to foster epithelial to mesenchymal transitioning of cancer cells via the activation of Snail, Twist and Zeb1 pathways. Interestingly, we also found that MGB1 confers breast cancer cell sensitivity to apoptosis and to various anti-cancer drug treatments.

Our study provides the first in dept molecular elucidation of MGB1 function in breast cancer processes. In summary, it extends our knowledge on the biology of breast cancer malignancy and gives us new tools to achieve new therapeutic and diagnostic strategies against this disease.

Citation Format: Nadia Picot, Annie-Pier Beauregard, Roxann Guerette, Stéphanie Jean, Gilles A. Robichaud. Breast cancer malignancy and drug sensitivity are regulated by mammaglobin 1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 298. doi:10.1158/1538-7445.AM2013-298
Abstract 2984: Understanding the relationship between a common mutation within the SWI/SNF chromatin remodeling complex in lung cancer and treatment response.
Authors: McNulty Maureen , Kirste Simon , Hu Jinwei , Singhal Udit , Liu Ziyan , Stegmaier Petra , Dressler Daniel , Chakravarti Arnab , Bell Erica
Published: August 2014

SWI/SNF is a chromatin remodeling complex that plays important roles in transcriptional regulation, DNA replication, and DNA repair. BRG1, one of its catalytic ATPase subunits, is a tumor suppressor that is mutated in approximately 20-30% of non-small cell lung cancers. To determine the clinical significance of BRG1 expression, non-small cell lung cancer data from The Cancer Genome Atlas was analyzed and showed that patients with stage II, high-BRG1 tumors have a higher overall survival than patients with stage II, low-BRG1 disease probably due to differences in genomic instability. BRG1’s involvement in multiple DNA repair pathways likely results in a decreased ability of BRG1-deficient tumor cells to repair damage, which underlies our hypothesis that DNA damaging therapies may be the best treatment for patients harboring deficient BRG1. In order to study the effects of DNA damage on BRG1-positive and BRG1-negative cells, we utilized ionizing radiation, ABT-888 (a PARP inhibitor), mitomycin C, and cisplatin to test BRG1’s role in double-strand break repair, base excision repair, nucleotide excision repair, and interstrand cross-link repair. Cell lines expressing a functional BRG1 (HeLa and H520) were approximately 2-4-fold less sensitive to radiation than cells without BRG1 (C33A and A427). Likewise, HeLa cells were about 4-fold less sensitive to cisplatin, a DNA crosslinking agent, than were C33A cells. We will present data from these assays in a panel of BRG1-positive and BRG1-negative non-small cell lung cancer cell lines, and in BRG1 knockdown and knock-in models. To ascertain the effect of BRG1 loss on the expression of genes involved in related pathways, BRG1-positive and BRG1-negative HeLa cells were analyzed with RT-PCR arrays. We found statistically significant dysregulation of genes involved in several pathways, including chromatin remodeling (SMARCA2, SMARCB1, and INO80) and DNA repair (XRCC4, CDK7, GTF2H1, and GTF2H2). We are also using RT-PCR arrays to study the panel of BRG1-positive and BRG1-negative cell lines. Elucidating novel roles for BRG1 in DNA repair pathways and determining its molecular targets will help identify the best treatment strategy for patients lacking functional BRG1, and may have the potential to increase the life expectancy of lung cancer patients.

Citation Format: Maureen McNulty, Simon Kirste, Jinwei Hu, Udit Singhal, Ziyan Liu, Petra Stegmaier, Daniel Dressler, Arnab Chakravarti, Erica Bell. Understanding the relationship between a common mutation within the SWI/SNF chromatin remodeling complex in lung cancer and treatment response. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2984. doi:10.1158/1538-7445.AM2013-2984
Abstract 2988: DOK2 suppression by methylation induces platinum resistance via suppression of apoptosis in ovarian cancer cells.
Authors: Lum Elena , Vigliotti Michele , Banerjee Nilanjana , Cutter Noelle , Kamalakaran Sitharthan , Wrzeszczynski Kazimierz , Khan Sohail , Dimitrova Nevenka , Levine Douglas , Lucito Robert
Published: August 2014

Ovarian cancers are highly heterogeneous and while chemotherapy is the preferred treatment, many patients are intrinsically resistant or quickly develop resistance. Furthermore, all tumors that recur will become resistant. Recent evidence suggests that epigenetic deregulation may be a key factor in the onset and maintenance of chemoresistance. To examine the ovarian epigenome, we first analyzed a set of 43 primary ovarian tumors and 9 normal ovarian samples. Since therapy response is a significant issue for ovarian cancer patients we analyzed the epigenetic differences that segregate with platinum response. We then associated expression data to identify genes with expression changes potentially altered by promoter methylation to generate a list of candidate platinum resistance genes. Next, we developed a tissue culture carboplatin resistance screen to determine which candidates functionally affect resistance. The screen utilized pools of shRNAs of the candidate genes to identify genes that when repressed allowed survival from carboplatin treatment, in order to validate that our epigenetics screen identified genes involved in resistance. Of the genes identified in the screen we further characterized one gene, docking protein 2 (DOK2), an adapter protein downstream of tyrosine kinase, to determine if we could elucidate what mechanism was used to increase resistance. Our analysis determined that loss of DOK2 decreased the level of apoptosis in response to carboplatin. In addition, we determined that in cells with reduced DOK2, the level of anoikis was decreased, a mechanism of possible importance in ovarian cancer where there is a large number of cells floating in ascites. Functional analysis of the DOK2 genes ability to affect resistance validates this approach to finding genes involved in carboplatin resistance.

Citation Format: Elena Lum, Michele Vigliotti, Nilanjana Banerjee, Noelle Cutter, Sitharthan Kamalakaran, Kazimierz Wrzeszczynski, Sohail Khan, Nevenka Dimitrova, Douglas Levine, Robert Lucito. DOK2 suppression by methylation induces platinum resistance via suppression of apoptosis in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2988. doi:10.1158/1538-7445.AM2013-2988
Abstract 2992: Stringent analysis of large-scale siRNA screens identifies a kinase fusion oncogene.
Authors: Ruefli-Brasse Astrid A. , Hart Cynthia , Fajardo Liza , Swearingen Elissa , Wong Linda , Sakamoto Doreen , Hale Kari , Ge Huanying , McDowell Jeffery , Ramachandran Bharath , Cosgrove Elissa , Watson J.E.Vivienne , Ragan Seamus , Ragan Seamus , Kassner Paul , Kassner Paul , Quon Kim
Published: August 2014

The interpretation of RNAi knockdown experiments can be challenging due to the extensive off-target effects exhibited by siRNAs and shRNAs. This has led to the reporting of results that cannot be independently verified, or to the identification of putative cancer targets that are insufficiently robust to support industrial drug discovery efforts. The problems associated with off target effects are particularly acute in high throughput RNAi screens, whose results often differ wildly between laboratories using different experimental platforms. As a result, the validity of published large-scale RNAi screens has recently been called into question.

Here we report results from large scale RNAi screens to identify genes essential for the proliferation or survival of cancer cells. To overcome the inherent liabilities of RNAi screens, we have increased the experimental power by using typically 8, and up to 20 RNAi triggers per gene, and have developed novel statistical algorithms that exploit this increased power. Importantly, this approach allows the calculation of a false discovery rate (FDR) that accurately predicts the ability of results to be confirmed by independent labs using independent reagents, solving a key issue of such screens. Using a stringent FDR cutoff of 5%, known essential genes and driver oncogenes such as KRAS, ERBB2 and BRAF are readily identified, demonstrating the robustness and power of the method. We have also identified a number of novel cancer targets. We present the identification and genetic and pharmacological validation of one of these targets, a kinase present as a fusion partner in multiple fusion oncogenes in primary tumors. Our data demonstrate that large-scale RNAi screens can be a robust and independently verifiable tool to uncover previously unknown cancer vulnerabilities which have yet to be characterized despite extensive genomic sequencing efforts.

Citation Format: Astrid A. Ruefli-Brasse, Cynthia Hart, Liza Fajardo, Elissa Swearingen, Linda Wong, Doreen Sakamoto, Kari Hale, Huanying Ge, Jeffery McDowell, Bharath Ramachandran, Elissa Cosgrove, J.E.Vivienne Watson, Seamus Ragan, Seamus Ragan, Paul Kassner, Paul Kassner, Kim Quon. Stringent analysis of large-scale siRNA screens identifies a kinase fusion oncogene. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2992. doi:10.1158/1538-7445.AM2013-2992
Abstract 2996: Systematic functional identification of driver mutations for individual ovarian cancers.
Authors: Mori Seiichi , Fujiwara Takeshi , Yokomizo Takako , Iwase Haruko , Kato Kazuyoshi , Nakashima Yukie , Kawaguchi Tokuichi , Noda Tetsuo
Published: August 2014

Although epithelial ovarian cancer is an intractable cancer and the overall five-year survival rate is approximately 40 to 50%, clinically effective molecular-targeted therapeutics to treat ovarian cancer has not been available thus far. Recent progress of massively parallel sequencing technologies has been enabling us to identify substantial number of genomic aberrations including somatic mutations, indels and translocations in variable cancer types exemplified by epithelial ovarian cancer and non-small cell lung cancer. However, it is still very difficult to identify a ‘driver’ gene, which is functionally relevant for cancer growth, from hundreds of mutations that are usually found from a single cancer sample of an individual patient. An in vitro cultured cell line, which is originated from a clinical sample, can be utilized to assess functions of the genes that are identified to have somatic aberrations in the paired clinical tumor. We have been establishing multiple ovarian cancer cell lines derived from clinical samples and analyzing pairs of the cell lines and clinical samples with exon-capture sequencing. The genes, which are somatically mutated and shared in a pair of cell line and clinical sample, are targeted with RNA interference in the cell line, and subsequently the effect on cell proliferation and survival is examined. As such, a driver mutation for an individual cancer can be identified by a set of experimental perturbations. Identified driver mutations would be then validated with publicly available databases and with clinical archive samples. We believe this approach can increase an opportunity to identify molecular targets in ovarian cancer therapeutics.

Citation Format: Seiichi Mori, Takeshi Fujiwara, Takako Yokomizo, Haruko Iwase, Kazuyoshi Kato, Yukie Nakashima, Tokuichi Kawaguchi, Tetsuo Noda. Systematic functional identification of driver mutations for individual ovarian cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2996. doi:10.1158/1538-7445.AM2013-2996
Abstract 300: CTGF is overexpression in asbestos-induced malignant mesothelioma and promotes beta-catenin-T-cell factor/Lef signaling.
Authors: Jiang Li
Published: August 2014

It is a well-known fact that malignant mesothelioma is primarily caused by the exposure to asbestos. Due to the widespread use of asbestos, cases of malignant mesothelioma are increasing at a dramatic rate. Histopathological examinations have revealed different subtypes of malignant mesothelioma. Based on cellular morphology, it is classified into epithelial (EM), sarcomatous (SM) and biphasic (BM) (containing a mixture of epithelial and sarcomatous cells) subtypes. Although sarcomatous mesothelioma is not as common as epithelial mesothelioma, it is recognized as the most aggressive type of malignant mesothelioma and patients diagnosed with this type of mesothelioma usually have a remarkably short survival period. Driven by the interest to know what are the molecular mechanisms underlying the relatively more aggressive phenotype of sarcomatous mesothelioma, we have initiated our study.

From the microarray results, we have singled out a candidate gene (connective tissue growth factor, CTGF) whose expression is significantly increased in SM samples compared to the EM and normal control. We found CTGF regulated mesothelioma cells, especial SM cells proliferation, migration, invasion via activating β-catenin-T-cell factor/Lef signaling pathway. Mesothelioma cells decreased proliferation, migration, invasion after knockdown CTGF. We injected mesothelioma cells or CTGF-knockdown mesothelioma cells into nude mice. The tumor size of CTGF-knockdown mesothelioma injection is significant smaller than non-treated mesothelioma, especially in SM. We also found a causal association between high CTGF expression and the poor prognosis of sarcomatous mesothelioma and that the underlying molecular mechanism is related to the promotion of EMT (epithelial-mesenchymal transition) by CTGF. Many EMT regulators including Slug, Snail and Twist have been activated when over-expressed CTGF in EM. Conversely, the MET also is found when knockdown CTGF in SM.

Our study is expected to have a great impact by offering novel therapeutic targets which are urgently needed at present. Our preliminary data appears to be very promising and we are highly confident that through the manipulation of our candidate gene, we will be able to at least greatly reduce the malignancy of sarcomatous subtype of mesothelioma and extend patient's lifespan, if not fully cure it.

Citation Format: Li Jiang. CTGF is overexpression in asbestos-induced malignant mesothelioma and promotes beta-catenin-T-cell factor/Lef signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 300. doi:10.1158/1538-7445.AM2013-300
Abstract 3001: Aberrantly up-regulated synaptotagmin-like 2 (SYTL2) promotes tumorigenesis of ovarian cancer cells .
Authors: Kim Sung-hoon , Mok Samuel C. , Wei Wei , Vinod Vathipadiekal , Mohapatra Gayatry , Birrer Michael
Published: August 2014

Epithelial ovarian cancer is the fifth leading cause of cancer-related death among woman and has the highest case-fatality rate among gynecologic cancers. Recently, microarray based high-throughput studies have characterized the genomics of epithelial ovarian cancer. These studies have revealed extensive DNA copy number alterations with large areas of gene amplification. There are however little data identifying the genes critical for the clinicopathologic characteristics of this tumor. Here, we describe an analysis of 11q14 amplicon which has been found in 25% of serous ovarian cancers. Among the genes in the 11q14 amplicon we validate an increased DNA copy number of SYTL2 in patient samples and show that this gene has the strongest negative prognostic power. Ectopic overexpression of SYTL2 in ovarian cancer cell lines A2780 and A224 showed increases in cell proliferation, colony formation, migration and invasion in vitro. In nude mice xenograft models, SYTL2 expression in A2780 cells resulted in increased tumor formation. SYTL2 may act through a novel mechanism by enhancing the secretion of Wnt-3a while having minimal effects on its expression. In summary, our data suggest SYTL2 has oncogenic effcst in ovarian cancer and may contribute to the progression of the disease.

Citation Format: Sung-hoon Kim, Samuel C. Mok, Wei Wei, Vathipadiekal Vinod, Gayatry Mohapatra, Michael Birrer. Aberrantly up-regulated synaptotagmin-like 2 (SYTL2) promotes tumorigenesis of ovarian cancer cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3001. doi:10.1158/1538-7445.AM2013-3001
Abstract 3009: Identification of cancer specific ERG target gene signatures.
Authors: Mohamed Ahmed , Ravindranth Lakshmi , Katta Shilpa , Tan Shyh-Han , Chen Yongmei , Petrovics Gyorgy , Dobi Albert , Srivastava Shiv
Published: August 2014

Introduction and Objective: ETS Related Gene (ERG) is aberrantly expressed in more than two-third of prostate cancers (CaP) due to gene fusions. CaP specific TMPRSS2- ERG fusion is a promising biomarker and potential therapeutic target. Cancer associated expression of ERG have also been described in several cancers. However, the precise molecular mechanisms underlying ERG induced oncogenic process in specific cancers are not clearly understood. Identification and thorough characterization of downstream target genes may aid to design effective therapeutic strategies for ERG expressing cancers. In this study, we have identified cancer specific ERG target gene signature of ERG expressing cancer cells that may be utilized as therapeutic targets for the treatment of ERG expressing CaP patients.

Methods: Illumina BeadChips microarray analysis was performed to profile ERG specific RNA signature from ERG positive tumor cell lines and normal ERG expressing endothelial cells transfected with either ERG specific siRNA or non-targeting siRNA. Functional gene clustering and Venn diagram analysis was employed to show the overlap of the common and unique gene sets regulated by ERG in these cells. Gene expression was analyzed by Western blot, QRT-PCR, and immunofluorescence assays.

Results: Biological function based gene clustering analysis identified two distinct gene signatures (normal and cancer specific) regulated by ERG. Knockdown of ERG in malignant cells resulted in overexpression of differentiation markers such as MSMB, KLK3, SLC45A3, NKX3.1, and NOV4 in prostate cancer (VCaP cells), and APC, DCC, CTNNB1, and NKD1 in colon cancer (COLO320 cells). In contrast, knockdown of ERG in normal endothelial-derived HUVEC cells resulted in the repression of differentiation markers such as CDH5, ANGEL2, and TIE1.

Conclusions: We identified tumor specific ERG target gene signature using normal and various cancer cell lines. This study confirmed the cell context dependent role of ERG in tumor and in normal endothelial cells. These observations have the potential to enhance our understanding of ERG biological functions thereby facilitate the design of suitable therapeutic mechanism.

Citation Format: Ahmed Mohamed, Lakshmi Ravindranth, Shilpa Katta, Shyh-Han Tan, Yongmei Chen, Gyorgy Petrovics, Albert Dobi, Shiv Srivastava. Identification of cancer specific ERG target gene signatures. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3009. doi:10.1158/1538-7445.AM2013-3009
Abstract 3010: Allele-specific loss of OSGIN1 associates with the progression of hepatocellular carcinoma.
Authors: Liu Ming , Li Yan , Chen Leilei , Chan Tim , Guan Xin-Yuan
Published: August 2014

Hepatocellular carcinoma (HCC) is one of the most frequent human malignances worldwide, which causes more than half a million death every year. Most human cancers are characterized by genomic instability. Chromosomal region gains or losses occur during cancer progression and high incidence of allelic imbalance is often observed in cancer patients. The risk alleles were usually overrepresented in cancer cells due to selective retention or amplification. In this report, we found a novel nucleotide variation at the coding region of the gene OSGIN1. Allele imbalance of OSGIN1 was frequently observed in a cohort of HCC patients, and the wild type allele was preferentially lost in the tumor tissue. Functional assays revealed that OSGIN1 has strong tumor suppressive function and associated with chemoresistance of HCC cell lines. The variant allele was less effective in inducing apoptosis than the wild type allele. This functional variation may be due to their different ability to localize to mitochondria. Further more, we found HCC patients carrying the variant allele have poorer prognosis than the wild type carriers. These findings suggested that OSGIN1 might be an important tumor suppressor gene in the progression of HCC and the retention of the variant allele might be a risk factor for HCC.

Citation Format: Ming Liu, Yan Li, Leilei Chen, Tim Chan, Xin-Yuan Guan. Allele-specific loss of OSGIN1 associates with the progression of hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3010. doi:10.1158/1538-7445.AM2013-3010
Abstract 3014: Implication of ΔNp73 isoform in drug resistance.
Authors: Soldevilla Beatriz , Rodríguez Marta , San Millán Coral , Bonilla Félix , Domínguez Gemma
Published: August 2014

Background: Aberrant expression of ▵Np73 has been associated with shorten cancer patient survival. We evaluate here whether this event could be due to the induction of drug resistance mechanisms.

Experimental Design: HCT116 colon cancer cell line transfected with a vector containing ▵Np73 or a mock vector were treated with oxaliplatin and checked for the viability by MTT, Flow-citometry, TUNEL assay and Caspase-3 protein expression levels. Additionally, we determined in 77 colon cancer patients the expression of ΔNp73 and their putative target genes related with drug resistance ABCB1, HMGB1 and CASP1 by quantitative real-time RT-PCR. Disease-free survival (DFS) and overall survival (OS) were examined in each patient.

Results: Ectopic expression of ▵Np73 significantly associated with a higher viability after oxaliplatin exposure. Positive correlations were observed between the expression levels of ▵Np73 variants and HMGB1. A trend was also observed for ABCB1. Overexpression of ΔNp73 isoforms predicted shortened OS (p = 0.05). High levels of ABCB1 and HMGB1 associated with shorter OS (p = 0.04 and p = 0.05, respectively). Multivariate analysis showed that, in addition to the tumor stage, ABCB1 and HMGB1 had independent relationships with OS (p = 0.008).

Conclusions:

The association of the upregulation of ▵Np73 with higher cancer cell viability after oxaliplatin treatment along with the fact that it could trigger HMG1 and ABCB1 in vivo, supports the hyphotesis that the shorten survival observed in those cancer patients showing overexpression of ▵Np73 could be due to the induction of drug resistance processes by this isoform.

Citation Format: Beatriz Soldevilla, Marta Rodríguez, Coral San Millán, Félix Bonilla, Gemma Domínguez. Implication of ΔNp73 isoform in drug resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3014. doi:10.1158/1538-7445.AM2013-3014
Abstract 3021: Mechanistic insight into MIG6 phosphorylation-mediated inhibitory effect on active EGFR.
Authors: Kim Kihong , Zhang Yi , Choi Kyusam , Do Ingu , Meyerson Matthew , Cho Jeonghee
Published: August 2014

Genomic alteration of Epidermal Growth Factor Receptor (EGFR) plays a crucial role in pathogenesis of various types of cancer. By a systematic quantitative phosphoproteomic approach (iTRAQ), we found that tyrosine phosphorylation occurs within segment 2 of MIG6, a negative feedback regulator of EGFR, in the cells transformed by lung-cancer derived EGFR mutants. We further confirmed this MIG6 phosphorylation is directly induced by activated EGFR by in vitro kinase assay with purified proteins followed by immunoblotting with phospho-specific antibody. Importantly, we also found that MIG6 phosphorylation seems to play a crucial role in its inhibitory role of EGFR activation. While wild-type MIG6 was able to completely inhibit the enzymatic activation of oncogenic mutant EGFR and subsequent cellular transformation, phosphorylation defective MIG6 mutants were unable to exert the same effects. Moreover, consistent with its functional role, we found that phosphorylation defective MIG6 failed to interact with mutant EGFR by co-immunoprecipitation assay, suggesting that the phosphorylation on MIG6 is required for interaction between MIG6 and EGFR. Taken together, we propose a working model that phosphorylation on segment 2 of MIG6 is directly induced by active EGFR, promoting tight interaction of MIG6 segment 2 and EGFR active site and subsequently blocking the enzymatic activity of EGFR.

Citation Format: Kihong Kim, Yi Zhang, Kyusam Choi, Ingu Do, Matthew Meyerson, Jeonghee Cho. Mechanistic insight into MIG6 phosphorylation-mediated inhibitory effect on active EGFR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3021. doi:10.1158/1538-7445.AM2013-3021
Abstract 3030: Regulation of EGFR in pancreatic cancer.
Authors: Halbrook Christopher J. , Takeuchi Kenneth K. , Crawford Howard C.
Published: August 2014

EGFR and EGFR ligand upregulation has been widely seen in both human chronic pancreatitis and pancreatic ductal adenocarcinoma (PDA) for many years; however the role of EGFR in PDA was poorly understood. Recently we and others have shown that genetic ablation of EGFR effectively prevents both spontaneous and cerulein induced tumorigenesis in the pancreas of the LSL-KrasG12D/+;Ptf1aCre/+ (KC) mouse model of pancreatic tumorigenesis. Pockets of EGFR upregulation localized in single acini of KC mice appear to precede pancreatic intraepithelial neoplasia (PanIN) formation in spontaneous tumorigenesis, coupled with a distinct spatially localized immune response. Ubiquitous EGFR upregulation is observed throughout the pancreas upon induction of pancreatitis in wild-type (WT) and KC mice, returning to normal levels in WT mice but continuing to increase along with the widespread tumorigenesis in cerulein treated KC mice. Comparison of cerulein treated KC mice with the EGFRf/f;LSL-KrasG12D/+;Ptf1aCre/+ mice (EKC), which are largely resistant to cerulein induced pancreatitis, offers an opportunity to identify signals in the KC mouse which could be responsible for the upregulation of EGFR and subsequent tumorigenesis.

Citation Format: Christopher J. Halbrook, Kenneth K. Takeuchi, Howard C. Crawford. Regulation of EGFR in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3030. doi:10.1158/1538-7445.AM2013-3030
Abstract 3037: Mer receptor tyrosine kinase is a novel therapeutic target in melanoma.
Authors: Schlegel Jennifer , Sambade Maria , Sather Susan , Moschos Stergios , Tan Aik-Choon , Winges Amanda , DeRyckere Deborah , Carson Craig C. , Trembath Dimitri G. , Tentler John J. , Eckhardt Gail , Kuan Pei-Fen , Hamilton Ronald L. , Duncan Lyn M. , Miller C. Ryan , Nikolaishvili-Feinberg Nana , Midkiff Bentley R. , Wang Xiaodong , Liu Jing , Zhang Weihe , Yang Chao , Frye Stephen V. , Earp H. Shelton , Shields Janiel , Graham Douglas K.
Published: August 2014

Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. While recent therapeutic advances have prolonged patient survival, prognosis remains dismal. Mer is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that Mer expression correlates with disease progression. Mer expression was highest in metastatic melanomas followed by primary melanomas whereas the lowest expression was observed in nevi. In addition, over 50% of melanoma cell lines overexpressed Mer compared to normal human melanocytes, however overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the Mer ligand Gas6 resulted in activation of several downstream signaling pathways including MAPK/ERK, PI3K/Akt, and Jak/STAT. Mer inhibition via shRNA reduced Mer-mediated downstream signaling, reduced colony formation by up to 59% (p<0.05) and diminished tumor volume by 60% (p<0.05) in a human melanoma xenograft murine model. Treatment of melanoma cells with UNC1062, a novel Mer-selective small molecule tyrosine kinase inhibitor, reduced activation of Mer-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar and inhibited invasion of melanoma cells. In addition, Mer inhibition synergized with mutant BRAF inhibition in signaling and apoptosis assays. This work establishes Mer as a therapeutic target in melanoma and provides rationale for the continued development of Mer-targeted therapies.

Citation Format: Jennifer Schlegel, Maria Sambade, Susan Sather, Stergios Moschos, Aik-Choon Tan, Amanda Winges, Deborah DeRyckere, Craig C. Carson, Dimitri G. Trembath, John J. Tentler, Gail Eckhardt, Pei-Fen Kuan, Ronald L. Hamilton, Lyn M. Duncan, C. Ryan Miller, Nana Nikolaishvili-Feinberg, Bentley R. Midkiff, Xiaodong Wang, Jing Liu, Weihe Zhang, Chao Yang, Stephen V. Frye, H. Shelton Earp, Janiel Shields, Douglas K. Graham. Mer receptor tyrosine kinase is a novel therapeutic target in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3037. doi:10.1158/1538-7445.AM2013-3037
Abstract 3039: Protein kinase C-δ mediated KIT recycling in colon cancers.
Authors: Park Mi Sun , Kim Won Kyu , Song Meiying , Kim Hoguen
Published: August 2014

Abnormal signals through the receptor tyrosine kinase (RTK) moieties are important in colon cancer both for tumorigenesis and drug targets. Wild type KIT, one of the RTKs, had been reported to be highly expressed in some colon cancers, however little is known about the functional aspects and roles of stem cell factor (SCF) dependent activation in colon cancers. We characterized colorectal cancers with KIT expression and analyzed the biological alterations of KIT after stimulation of SCF and its impact on the downstream signals. We identified wild type KIT is expressed in some colon cancer cell lines, activated by SCF, and subsequently activated AKT and ERK signaling pathways. We also demonstrated that KIT expression was gradually decreased after SCF stimulation due to lysosomal degradation of activated KIT. We found PKC activation rescues KIT by inhibiting ubiquitination of KIT after SCF treatment. Among the various types of PKCs, PKC-δ directly bound to wild type KIT and rescued SCF-KIT complexs. Additionally, we demonstrated that KIT and PKC-δ are concomitantly activated in the colon cancer tissues with wild type KIT expression, and the expression of KIT is correlated with poor survival. Based on these findings we concluded that downstream signal activation after SCF-KIT binding is continuously accomplished by the PKC-δ activation and subsequent KIT recycling. These signal pathways induced by SCF and PKC-δ mediated KIT recycling play important roles in colon cancer progression.

Citation Format: Mi Sun Park, Won Kyu Kim, Meiying Song, Hoguen Kim. Protein kinase C-δ mediated KIT recycling in colon cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3039. doi:10.1158/1538-7445.AM2013-3039
Abstract 3065: The role of miR-18b in MDM2-p53 pathway signaling and melanoma progression.
Authors: Dar Altaf A. , Majid Shahana , de Semir David , Bezrookove Vladimir , Nosrati Mehdi , Kashani-Sabet Mohammed
Published: August 2014

MicroRNAs regulate gene expression and are differentially expressed in various tumors. While p53 is inactivated by point mutations in many tumors, melanomas rarely harbor mutations in the p53 gene. Here we report the biological role of miR-18b in melanoma via targeting the MDM2-p53 pathway. Expression of miR-18b was significantly suppressed in melanoma specimens and cell lines by virtue of hypermethylation, and was re-induced in melanoma cell lines following 5-AZA-deoxycytidine treatment. Lower levels of miR-18b expression in primary melanoma were associated with significantly reduced overall survival. MDM2 was identified as a target of miR-18b action, as miR-18b significantly suppressed the luciferase activity of a reporter plasmid containing the 3’UTR sequence of MDM2 complementary to the miR-18b seed sequence. Induction of miR-18b expression in melanoma cells resulted in reduced MDM2 expression, upregulation of p53 as well as PUMA and p21, and suppression of the anti-apoptotic BCL2 and BCL-XL genes, resulting in significant suppression of melanoma cell proliferation. The effects of miR-18b overexpression on the p53 pathway and on melanoma cell growth were reversed by MDM2 overexpression. Stable overexpression of miR-18b produced potent tumor suppressor activity, as evidenced by suppressed melanoma cell proliferation, induction of apoptosis, and reduced tumor cell growth in vivo. In addition, miR-18b overexpression suppressed melanoma cell migration and invasiveness, and reversed epithelial-to-mesenchymal transition in melanoma cell lines. Our results demonstrate a novel role for miR-18b as a tumor suppressor in melanoma, identify the MDM2-p53 pathway as a target of miR-18b action, and suggest miR-18b overexpression as a novel strategy to reactivate the p53 pathway in human tumors.

Citation Format: Altaf A. Dar, Shahana Majid, David de Semir, Vladimir Bezrookove, Mehdi Nosrati, Mohammed Kashani-Sabet. The role of miR-18b in MDM2-p53 pathway signaling and melanoma progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3065. doi:10.1158/1538-7445.AM2013-3065
Abstract 3066: Comprehensive interactome of embryonic transcription factors, mitogenic stresses and miRNAs involved in breast tumorigenesis.
Authors: Ruiz Emmanuelle , Courtois-Cox Stephanie , Gras Baptiste , Veron Amélie , Ansieau Stephane , Puisieux Alain , MOYRET-LALLE Caroline
Published: August 2014

Epithelial-to-Mesenchymal Transition (EMT) is associated with tumour initiation and progression. EMT is associated with a profound genetic reprogramming including reactivation of embryonic transcription factors (ETFs) and severe changes in miRNA expression. Cross-regulation between EMT-inducers and miRNAs was previously described like the Zeb1-miR-200 feed-back loop. Our goal is to determine whether specific expression networks between ETFs, miRNAs and mitogenic stresses exist in tumors, and represents a driving force towards transformation in breast tumorigenesis.

We performed oncogenic cooperation assays in immortalized human mammary epithelial cells (HMEC-hTert) by using an EMT-inducer expression library in combination with various mitogenic stresses (activated form of beta-catenin, EGFR, shPTEN, shP53). As recently demonstrated in the laboratory, these combinations were efficient in transforming human cells, as assessed by soft-agar colony assay. EMT-inducer expression was next analyzed in a large number of colonies to evaluate a potential specificity of EMT-inducers according to the mitogenic insult.

We found that expression of Slug was specifically enriched in colonies generated when EGFR is highly expressed, whereas Zeb1 expression was selected following PTEN depletion, p53 inactivation and beta-catenin activation. FoxC2 expression was enriched in cells expressing the activated form of beta-catenin. The prediction of specificity of the identified combinations is currently being validated by bioinformatics tools. In parallel, we developed an in silico approach utilizing predictive algorithms to identify novel miRNAs targeting embryonic transcription factors. We have identified two novel miRNAs able to regulate EMT inducers and we have validated their down-regulation in a panel of basal B breast cancer cell lines. We have also found new feedback loops between these two miRNA and some ETFs. The interplay between miRNAs, mitogenic proteins and EMT inducers will be further examined by assessing their expression in a large cohort of Basal B/ Claudin-low human breast tumours, a recently identified subtype displaying a mammary stem cell signature and EMT features.

Citation Format: Emmanuelle Ruiz, Stephanie Courtois-Cox, Baptiste Gras, Amélie Veron, Stephane Ansieau, Alain Puisieux, Caroline MOYRET-LALLE. Comprehensive interactome of embryonic transcription factors, mitogenic stresses and miRNAs involved in breast tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3066. doi:10.1158/1538-7445.AM2013-3066
Abstract 3067: microRNAS implicated in the regulation of NF-κB activity in glioma tumors.
Authors: Haemmig Stefan , Vassella Erik
Published: August 2014

Gliomas are among the most prognostically discouraging neoplasia in human and there is basically no curative option for glioma patients. Nuclear factor κB (NF-κB) is a key effector associated with temozolomide (TMZ) resistance in glioma tumours. This is at least in part due to down-regulation of TNFAIP3, which is a critical negative regulator of NF-κB activity. Downregulation of this gene is strongly associated with resistance to TMZ, but the molecular mechanism of dysregulation is largely unknown. We hypothesize that microRNAs that are implicated in the NF-κB pathway may be important regulators of resistance to alkylating agents in glioma tumours.

TNFAIP3 is a predicted target of brain-enriched microRNAs miR-125a and miR-125b. Consistent with these findings, luciferase activity assays of reporter plasmids containing miR-125a/b target sites revealed that TNFAIP3 is directly regulated by physiological concentrations of miR-125a/b. In addition, overexpression of miR-125a/b resulted in reduced mRNA and protein levels of TNFAIP3 and, as a consequence, also in a significant induction of NF-κB activity. Consistent with these findings, glioblastoma cells overexpressing these miRNAs showed a strong upregulation of the anti-apoptotic proteins Bcl-2 and BIRC3. Interestingly, this was significantly associated with resistance of glioma cells to TNFalpha- and TRAIL-induced apoptosis and also resistance to TMZ. A siRNA directed against TNFAIP3 and miR-125a/b gave rise to similar phenotypes suggesting that TNFAIP3 is a major target of miR-125a/b. In glioblastoma tissues, high level expression of either miRNA was highly correlated with nuclear localisation of NF-κB confirming that miR-125a/b are implicated in NF-κB signalling. Most remarkably, miR-125a/b expression was clearly associated with shorter overall survival of patients treated with TMZ, suggesting that these miRNAs are important predictors of response to therapy. However, we found no correlation between miR-125a/b levels and the MGMT methylation status.

In conclusion, miR-125a/b expression is a predictor of the patient's response to chemotherapy. Since miR-125a/b confers resistance to TMZ, antisense miR-125a/b might potentially be used in adjuvant therapy for the treatment of glioma tumours.

Citation Format: Stefan Haemmig, Erik Vassella. microRNAS implicated in the regulation of NF-κB activity in glioma tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3067. doi:10.1158/1538-7445.AM2013-3067