bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1918: The role of miR17-92 in the miRegulatory landscape of Ewing sarcoma
Authors: Schwentner Raphaela , Kauer Maximilian , Herrero-Martin David , Kovar Heinrich
Published: July 2016

MicroRNAs (miRNA) serve to fine-tune gene expression and thus play an important regulatory role in tissue specific gene networks. The identification and validation of miRNA target genes in a given tissue still poses a significant problem since the presence of a seed sequence in the 3′UTR of an mRNA and its expression modulation upon forced ectopic expression of the miRNA do not reliably predict regulation under physiological conditions. The chimeric oncoprotein EWS-FLI1 is the driving pathogenic force in ES. Recently, we reported on the EWS-FLI1 microRNA (miRNA) signature based on knockdown experiments in ES cell lines and on differential expression in primary tumors versus mesenchymal stem cells. Comparison of both datasets revealed the miR17-92 cluster to be the top EWS-FLI1 activated miRNA, which is one of the most potent oncogenic miRNAs. The whole cluster is among the small number of miRNAs down-regulated upon knockdown of EWS-FLI1 in multiple ES cell lines. We now report the use of a combination of AGO2 pull-down experiments by PAR-CLIP - to enrich and sequence RISC-associated RNAs - and of RNAseq upon miRNA depletion by ectopic sponge expression to identify the targetome of miR17-92 in Ewing sarcoma (ES) cells

We found a significant enrichment of PAR-CLIP hits for members of the miR-17-92 cluster in the 3′UTRs of genes up-regulated in response to mir-17-92 but not miR9 and 10b specific sponge expression. Among them, we consistently identified known (i.e. CTGF, MXD1, CYLD1) and a multitude of so far unknown targets of the oncomir-1 cluster in ES in three PAR-CLIP experiments. Interestingly, approximately a quarter of these genes annotate to the TGFB/BMP pathway, the majority mapping downstream of SMAD signaling.

Gene reporter assays using 3’UTRs of select novel candidate genes showed enhancement of luciferase activity upon transfection of a sponge specific to miR-17-92 and 20a, but not to unrelated miR-9 and 10b. Site directed mutagenesis of seed sequences in these 3’UTR confirmed the direct regulation of these genes by miR17-92. Current research focuses on the functional validation of TGFB pathway modulation by miR17-92. This study provides a paradigmatic approach to the comprehensive definition of the targetome for a defined group of miRNAs under physiological conditions.

This study was supported by the 7th framework program of the European Commission (FP7-259348, “ASSET”) and the Austrian Science Fund FWF (24708-B21)

Citation Format: Raphaela Schwentner, Maximilian Kauer, David Herrero-Martin, Heinrich Kovar. The role of miR17-92 in the miRegulatory landscape of Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1918.
Abstract 1925: Regulation of metabotropic glutamate receptor 5 expression by the miR-30 downregulation induced by the SDF-1/CXCR4 system in oral cancer cells
Authors: Kuribayashi Nobuyuki , Uchida Daisuke , Kinouchi Makoto , Koshiji Chikako , Izumi Sayaka , Hakata Kembun , Komiyama Yuske , Tsuchida Shuji , Hasegawa Tomonori , Kawamata Hitoshi
Published: July 2016

We have previously demonstrated that stromal cell-derived factor (SDF)-1/CXCR4 system enhances the metastases of oral cancer cells via induction of metabotropic glutamate receptor (mGluR) 5. However, the mechanism(s) of mGluR5 expression induced by the SDF-1/CXCR4 system are largely unknown. Recently, small non-coding RNAs, microRNAs (miRNAs) are involved in the metastatic process of several types of cancers. In this study, we examined the miRNA association involved in the mGluR5 expression using oral cancer cells, B88, which express functional CXCR4 and exhibit highly metastatic potentials. We examined the metastasis-related miRNAs in SDF-1 stimulated B88 cells by use of a miRNA microarray analysis. Consequently, we isolated miR-30 family which has predictive binding sites in 3’-UTR of mGluR5 mRNA in silico analysis. Among these miRNAs, we confirmed the downregulation of all miR-30 family in SDF-1 stimulated B88 cells by the real-time PCR analysis. Next, we transfected these miR-30 families in SDF-1 stimulated B88 cells. We confirmed the downregulation of mGluR5 by the miR-30a-5p and miR-30c-5p overexpression. These results indicated that SDF-1/CXCR4 system might regulate the metastases of oral cancer by the upregulation of mGluR5 via downregulation of miR-30 family.

Citation Format: Nobuyuki Kuribayashi, Daisuke Uchida, Makoto Kinouchi, Chikako Koshiji, Sayaka Izumi, Kembun Hakata, Yuske Komiyama, Shuji Tsuchida, Tomonori Hasegawa, Hitoshi Kawamata. Regulation of metabotropic glutamate receptor 5 expression by the miR-30 downregulation induced by the SDF-1/CXCR4 system in oral cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1925.
Abstract 1934: MicroRNA-383 located in frequently deleted chromosomal locus 8p22 regulates prostate cancer stem cell marker CD44
Authors: Bucay Nathan , Sekhon Kiran , Shahryari Varahram , Mitsui Yozo , Deng Guoren , Tabatabai Z. Laura , Majid Shahana , Yamamura Soichiro , Dahiya Rajvir , Tanaka Yuichiro , Saini Sharanjot
Published: July 2016

A major genomic alteration in prostate cancer (PCa) is frequent loss of chromosome (chr) 8p with a common region of loss of heterozygosity (LOH) at chr8p22 locus. Genomic studies implicate this locus in the initiation of clinically significant PCa and with progression to metastatic disease. However, the genes within this region have not been fully characterized to date. Here we demonstrate for the first time that a microRNA component of this region -miR-383- is frequently downregulated in prostate cancer, plays a critical role in determining tumor initiating potential and is involved in prostate cancer metastasis via direct regulation of CD44, a ubiquitous marker of PCa tumor initiating cells (TICs)/ stem cells. Expression analyses of miR-383 in PCa clinical tissues established that low miR-383 expression is associated with poor prognosis. Functional data suggests that miR-383 regulates PCa tumor initiating/ stem-like cells via CD44 regulation. Ectopic expression of miR-383 inhibited tumor initiating capacity of CD44+ PCa cells. Also, ‘anti-metastatic’ effects of ectopic miR-383 expression were observed in a PCa experimental metastasis model. In view of our results, we propose that frequent loss of miR-383 at chr8p22 region leads to tumor initiation and prostate cancer metastasis. Thus, we have identified a novel finding that associates a long observed genomic alteration to PCa stemness and metastasis. Our data suggests that restoration of miR-383 expression may be an effective therapeutic modality against PCa relapse and metastasis. Importantly, we identified miR-383 as a novel PCa diagnostic biomarker with potential that outperforms that of serum PSA.

Citation Format: Nathan Bucay, Kiran Sekhon, Varahram Shahryari, Yozo Mitsui, Guoren Deng, Z. Laura Tabatabai, Shahana Majid, Soichiro Yamamura, Rajvir Dahiya, Yuichiro Tanaka, Sharanjot Saini. MicroRNA-383 located in frequently deleted chromosomal locus 8p22 regulates prostate cancer stem cell marker CD44. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1934.
Abstract 1943: A microRNA signature in urinary exosomes for diagnosis of prostate cancer
Authors: Blondal Thorarinn , Rasmussen Anne I. , Thomsen Anni R. , Borre Michael , Fredsøe Jacob , Andreasen Ditte , Falck Ørntoft Torben , Sørensen Karina D. , Mouritzen Peter
Published: July 2016

MicroRNAs constitute a class of small cellular noncoding RNAs (typically 19-23 nt) which function as post-transcriptional regulators of gene expression. The involvement of microRNA in both normal development as well as disease is well established and microRNAs are considered important biomarkers in several diseases including cancer. Surprisingly cell free microRNAs are also found in several biofluids where they have been shown to be very stable, most likely because they are protected from degradation by protein complexes and other particles including exosomes.

Exosomes are nanovesicles secreted into the extracellular environment by a wide range of cell types under normal and pathological conditions. As the profile of exosomal microRNAs may be a fingerprint of the releasing cell type and because they are released in easily accessible body fluids such as blood and urine, their microRNA content holds potential as biomarkers for early detection of malignancy.

We have developed different technologies enabling the detection of prostate cancer microRNA biomarkers in exosomes derived from urine from non-prostate massaged men. The first of these technologies is a highly sensitive LNA™-based qPCR platform for microRNA detection, which enables profiling in biofluids where microRNA levels are extremely low. At this point we have already applied this platform on thousands of biofluid samples including serum/plasma and urine to establish normal reference ranges for circulating microRNAs as well as to identify biomarkers of disease. The second technology is a simple exosome precipitation system which only requires low speed centrifugation to harvest urine exosomes.

Our developed technologies was applied to cell-free urine from three different cohorts including individuals with prostate cancer (PCa) and benign prostate hyperplasia, to identify several differentially regulated microRNAs in urine from PCa bearing individuals. PCa specific signatures were obtained by different combinations of these differentially regulated microRNAs. In conclusion, cell free urine samples holds potential as a liquid biopsy source for exosomal microRNA markers in prostate cancer. Data showing that small miRNA signatures (down to two miRNAs) hold strong diagnostic potential in PCa will be presented.

Citation Format: Thorarinn Blondal, Anne I. Rasmussen, Anni R. Thomsen, Michael Borre, Jacob Fredsøe, Ditte Andreasen, Torben Falck Ørntoft, Karina D. Sørensen, Peter Mouritzen. A microRNA signature in urinary exosomes for diagnosis of prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1943.
Abstract 1951: Expression profiling, cellular and clinical implications of mitochondrial miRNAs in colorectal carcinomas
Authors: Ebrahimi Faeza , Gopalan Vinod , Lam Alfred
Published: July 2016

Aim: The aim of this study was to determine the expression profiling and cellular implications of mitochondrial miRNAs (Mitomirs) such asmiR-145, miR-15a, miR-335 and miR-126)in colorectal cancer tissues. Expression changes in matched metastatic tissues were also examined.

Method: Colorectal tissues from163 primary cancers, 96 lymph node, 48 metastatic, 40 adenoma and 42 non-neoplastic tissues were recruited from 389 patients. The expression of Mitomirs in tissues and colon cancer (SW-480, SW-48) and normal (FHC) cell lines were investigated by qRT-PCR. Functional assays including cell proliferation, cell cycle analysis and apoptosis assays were performed with an exogenous miR-126 mimic to detect its in-vitro changes on cancer cell biology. In addition, Western blot was used to detect the targets of miR-126.

Result: Expressions of Mitomirs were down-regulated in colorectal cancer tissues compared to non-tumour colorectal tissues. Higher expression of miR-145 was predominantly noted in colorectal cancers with tumour recurrence (P = 0.058). Expression of miR-335 was significantly reduced distal colorectum compared to proximal colon (P = 0.025). Reduced expression of miR-335 also showed significant correlation with occurrence of lymphovascular permeation (P = 0.019), and late T stage (P = 0.004) of colorectal cancer. Moreover, in microsatellite instability (MSI) positive adenocarcinomas, notable downregulation of miR-15a (P = 0.018) was observed and this reduced expression was significantly correlated with size of tumour (> 40 mm) (P = 0.014). Expression of miR-15a, and miR-145 was reduced in metastatic cell line (SW48). Overexpression of miR-126 in-vitro showed increased apoptosis, reduced cell proliferation and suppression of synthetic phase in cell cycle. Also, overexpression of miR-126 has induced reduced expression of BCL-2 protein and increased expression of P53 protein.

Conclusion: This study suggests that expression of Mitomirs significantly reduces in advanced stages of cancer and even in early stages in comparison to normal tissues. In vitro studies indicated that ectopic expression miR-126 has the capability of inducing apoptosis and reducing viability of cancers cells potentially via modulating its target partners such as BCL-2 and p53.

Citation Format: Faeza Ebrahimi, Vinod Gopalan, Alfred Lam. Expression profiling, cellular and clinical implications of mitochondrial miRNAs in colorectal carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1951.
Abstract 1966: Automated miRNA purification without the use of organic solvents
Authors: English Jami D. , Horejsh Doug , Moreland Chris , Urh Marjeta
Published: July 2016

MicroRNAs (miRNAs) are endogenous small non-coding RNAs 18-24 nucleotides in size that play important roles involving gene regulation associated with cancer, disease control and gene silencing. Because of the impact on disease progression, miRNA research is rapidly shifting towards biomarker discovery. Many of the commercially available miRNA purification methods involve organic extraction during pre-processing. Here, we describe a novel chemistry that enables total RNA purification including smaller RNA such as miRNA. This chemistry offers significant advantages with a simple automated workflow, no organic extraction and minimal pre-processing.

In this study, we successfully show purification of miRNAs from a range of solid and liquid sample types including frozen tissue, whole blood, plasma, exosomes, mammalian cell lines, saliva, bone and FFPE samples. Absorbance yield, absorbance ratios, RINs, and amplification of miRNA show equivalence with organic solvent-based purification methods. The miRNA studied included miR-21, let-7a, miR-141, and miR-125b, depending on starting sample type. Next generation sequencing (NGS) analysis shows equivalent sequence diversity when compared pairwise with a manual, organic pre-processing method.

This novel chemistry and method enables purification of small and large RNA from a range of sample types. The purified RNA has little or no detectable DNA contamination and does not require phenol extraction, thus improving safety. Also, the automation of this workflow decreases manual hands on time, saving scientist time and reducing the risk of RNase contamination.

Citation Format: Jami D. English, Doug Horejsh, Chris Moreland, Marjeta Urh. Automated miRNA purification without the use of organic solvents. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1966.
Abstract 1970: miRNA differential profiling between fibroblasts originated from breast cancer and normal adjacent tissue from benign disease
Authors: Rozenchan Patricia Bortman , de Carvalho Cristina V. , Bonetti Tatiana , Melo Carina , Brentani M Mitzi , Girão Manoel JBC
Published: July 2016

The importance of tumor-stromal cells interactions in breast tumor progression and invasion has been recognized; therefore, modifications in the stromal fibroblasts can play a significant role in overall cancer development and progression. Accumulating evidence indicates that epigenetic phenomena such as microRNAs are extremely important and were already recognized as key regulators of gene expression, as well as expression of these small RNAs has been linked to human disease, including those related to estrogen-dependent disorders and in cancer homeostasis. So, deciphering molecular alterations in breast tumor microenvironment such as miRNAs differential expression could lead not solely to target for therapeutic intervention as well as to novel early diagnosis markers. Stromal fibroblasts were isolated from four invasive breast carcinoma samples (CAFs) and from five women with benign breast disorders (NAFs). RNA was extracted from low-passage cultures and transformed into cDNA and used for the Human Breast Cancer miScript® miRNA PCR Array expression analysis on StepOne Plus Real Time. This Array profiles the expression of 84 mirRNAs known or predicted to alter their expression during breast cancer initiation or progression. We could find 05 differentially expressed miRNAs in CAFs in comparison to NAFs; 04 down-regulated: mir107 (fold: -1,56, p = 0,04), miR-125b-1 (fold:-1,83, p = 0,03), miR-200b (fold: -2, p = 0,02), miR-93 (fold: -2,25, p = 0,02) and 01 up-regulated: miR-424 (fold: 5,73, p = 0,01). After bioinformatics analysis, these miRNAs were predicted to regulate genes involved in important cancer related pathways like, Cyclins and Cell Cycle Regulation; EGF Signaling Pathway; MAPKinase Signaling Pathway and Apoptotic Signaling in Response to DNA Damage. All together, our results showed that there are important differences regarding miRNAs expression profiling between CAFs and NAFs that could be determinant for breast cancer tumorigenesis.

Citation Format: Patricia Bortman Rozenchan, Cristina V. de Carvalho, Tatiana Bonetti, Carina Melo, M Mitzi Brentani, Manoel JBC Girão. miRNA differential profiling between fibroblasts originated from breast cancer and normal adjacent tissue from benign disease. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1970.
Abstract 1975: The 3D chromatin structure of the PTHLH region comprises a dynamic hierarchical looping complex that approximates the protein-coding genes and facilitates promoter and enhancer promiscuity
Authors: Freeman Adam N. , Henderson Michael A. , Kapuscinski Miroslaw K. , Hopper John , Martin T John
Published: July 2016

Parathyroid Hormone-Like Hormone (PTHLH) is an important regulatory gene encoding the product Parathyroid Hormone-related Protein (PTHrP). Among many biological roles, it has been demonstrated to be essential to the development of multiple tissue-types, the regulation of foetal calcium levels, and the metastasis of breast cancer to bone. Its region has GWAS associations with breast cancer, breast size, height, type 2 diabetes mellitis, neural development, cardiac arrest, and several immunological phenotypes. They extend both up- and down-stream of PTHLH, however their driving molecular mechanisms remain obscure.

Analysis of datasets including Hi-C, ChIA-PET, IM-PET and ChIP-seq suggests PTHLH sits within a 1.3Mb Topologically Associating Domain (TAD) containing 4 other protein-coding genes (pc-genes) CCDC91, MRPS35, MANSC4 and KLHL42. PTHLH itself rests between two 220kb inducible sub-TADs that likely comprise regulatory archipelagos directed primarily at its regulation. There are several other inducible loops in the TAD.

All protein-coding genes within the TAD feature an Activated Chromatin Hub (ACH) nearby their 5’. The 3D-structure of the TAD appears to be supported by a system of permanent and dynamic tether points producing primary and inducible chromatin loops. These result in all pc-genes’ ACHs being 30 to 60kb from each other. RNA Pol II (RNAPII) ChIA-PET data suggests there is an inclusive hierarchical regulatory structure within the TAD, with substantial interaction between the ACHs of respective pc-genes that is likely facilitated by the 3D conformation.

Citation Format: Adam N. Freeman, Michael A. Henderson, Miroslaw K. Kapuscinski, John Hopper, T John Martin. The 3D chromatin structure of the PTHLH region comprises a dynamic hierarchical looping complex that approximates the protein-coding genes and facilitates promoter and enhancer promiscuity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1975.
Abstract 1988: MYB overexpression is associated with castration-resistance in prostate cancer
Authors: Srivastava Sanjeev K. , Bhardwaj Arun , Singh Seema , Arora Sumit , Tyagi Nikhil , Carter James E. , Singh Ajay P.
Published: July 2016

Clinical progression of prostate cancer (PCa) is characterized by a transition from castration-sensitive (CS) to castration-resistant (CR) phenotype. We previously demonstrated that MYB overexpression sustained the growth of PCa cells in androgen-depleted growth environment as well as promoted aggressive tumor phenotypes. Here, we examined its role in PCa progression and castration-resistance using appropriate mouse models and delineated underlying molecular mechanisms. Luciferase-tagged MYB-overexpressing (LNCaP-MYB) or -silenced (C4-2-shMYB) PCa cell lines along with their respective control sublines (LNCaP-Neo and C4-2-NTScr) were orthotopically implanted in nude mice and tumor growth was monitored by non-invasive bioluminescence imaging using Xenogen-IVIS optical system. Our data demonstrated significantly enhanced tumor growth in mice implanted with high MYB expressing (LNCaP-MYB and C4-2-NTScr) PCa cells as compared to low MYB expressing (LNCaP-Neo and C4-2-shMYB) cells. To study the role of MYB in castration-resistance, we castrated the mice after tumors from all groups reached comparable size as determined by in vivo imaging. Tumors in LNCaP-Neo and C4-2-shMYB groups exhibited drastic regression following castration. In contrast, only slight initial reduction in the tumor growth was reported in mice from LNCaP-MYB and C4-2-NTScr groups following castration, and they later regained tumor growth. Furthermore, Kaplan-Meier survival analysis revealed that the median survival of testis-intact (non-castrated) animals of LNCaP-Neo and C4-2-shMYB groups was significantly lower than that of castrated group mice. However, castration in high MYB-expressing tumor bearing mice could barely improve their median survival. Interestingly, MYB expression levels in orthotopic tumors correlated with serum PSA levels. Furthermore, castration of mice in LNCaP-Neo and C4-2-shMYB groups resulted in a rapid reduction in serum PSA levels; while it sustained and continued to rise in MYB overexpressing groups. In additional studies, we demonstrated that MYB interacts with androgen-receptor (AR) in prostate cancer cells, and facilitates the nuclear localization of AR in a ligand-independent manner. In essence, these results suggest a critical role of MYB in the CR progression of PCa.

Citation Format: Sanjeev K. Srivastava, Arun Bhardwaj, Seema Singh, Sumit Arora, Nikhil Tyagi, James E. Carter, Ajay P. Singh. MYB overexpression is associated with castration-resistance in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1988.
Abstract 199: Evaluation of multiple EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants using in vitro modeling
Authors: Hasegawa Hanako
Published: July 2016

Purpose:

We are able to use multiple EGFR-TKIs to treat patients with lung cancer harboring EGFR mutations. However, there is no clear guideline regarding which EGFR-TKIs should be used for which mutations. The purpose of this study is to establish an in vitro model to determine the “therapeutic window” of EGFR-TKIs against various types of EGFR mutations, including several EGFR exon 20 insertion mutations.

Experimental design:

We evaluated drug sensitivity and downstream signaling of human lung cancer cell lines harboring EGFR mutations (PC9, H3255, PC9-ER, BID007 and H1975) and Ba/F3 cells exogenously expressing mutated or wild type EGFR for the 1st (erlotinib), 2nd (afatinib), and 3rd (osimertinib and rociletinib) generation EGFR-TKIs using MTS assay, apoptosis assay and western blotting, simultaneously.

An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFRs.

Results:

The model of mutation specificity identified wide therapeutic windows of afatinib for exon 19 deletions and L858R and those of osimertinib and rociletinib for T790M positive mutations in human cell lines and Ba/F3 cells. In human cell lines and Ba/F3 cells harboring exon 19 deletions or L858R, afatinib showed the lowest IC50 and most potently inhibited the phosphorylation of EGFR, AKT, and ERK. Afatinib and 3rd generation EGFR-TKIs (osimertinib and rociletinib), effectively inhibited the cell growth and the phosphorylation of downstream signaling of EGFR in T790M positive cells. For EGFR exon 20 insertion mutations, osimertinib was potent and presented the most wide therapeutic window.

Conclusion:

This model will provide a preclinical rationale for proper selection of EGFR-TKIs against clinically-relevant EGFR mutations including EGFR exon 20 insertions.

Citation Format: Hanako Hasegawa. Evaluation of multiple EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants using in vitro modeling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 199.
Abstract 1990: Unique in vitro transcriptional activities of oncogenic Myb fusions identified in salivary gland adenoid cystic carcinoma
Authors: Frerich Candace A. , Ness Scott A.
Published: July 2016

Chromosomal translocations involving the MYB and NFIB (t(6;9)) genes are the sole recurrent cytogenetic aberration in Adenoid Cystic Carcinoma (ACC), a rare cancer that frequently manifests as salivary gland tumors. Since ACC has poor prognosis and treatment is accompanied by high morbidity, there is a need for additional investigation of this cancer with the goal of improving patient outcomes. Recurrent translocations create fusions involving the N-terminus of MYB, located on chromosome 6, and the C-terminus of NFIB, located on chromosome 9. Our lab also recently identified translocations involving the related MYBL1 gene fusing it to NFIB t(8;9) and RAD51B t(8;14) genes. Further, recent work in our lab demonstrated tumors with MYB or MYBL1 translocations have similar gene expression (1). The presence of recurrent translocations involving both the MYB proto-oncogene and the related MYBL1 gene suggests that the resulting novel fusion proteins are likely oncogenic drivers in these tumors. In order to directly address the transcriptional activities of the resulting fusion proteins MYB and MYBL1 fusion genes, identified in primary ACC tumors, were cloned and expressed in vitro. The fusion proteins transcriptional activities were examined using reporter gene assays and trancriptomics. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed to interrogate promoters bound by these proteins. Reporter genes specific for normal vs. oncogenic MYB variants have been constructed. These data demonstrate that the proteins predicted to be produced by MYB fusion genes after chromosomal translocation are transcriptionally active. Further, Myb fusion proteins bind and activate gene sets distinct from their wild type c-Myb and A-Myb counterparts. These data indicate Myb fusion proteins have unique transcriptional activities, perhaps underlying their ability to drive tumorigensis in ACC.

1. Brayer et al. Recurrent Fusions in MYB and MYBL1 Define a Common, Transcription Factor-Driven Oncogenic Pathway in Salivary Gland Adenoid Cystic Carcinoma. Cancer Discovery. In press.

Citation Format: Candace A. Frerich, Scott A. Ness. Unique in vitro transcriptional activities of oncogenic Myb fusions identified in salivary gland adenoid cystic carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1990.
Abstract 1991: Mutant GATA3 actively promotes the growth of normal and malignant mammary cells
Authors: Chandiramani Natasha , Peterson Esther , Kenny Paraic A.
Published: July 2016

GATA3 is a transcription factor expressed in luminal breast epithelial cells and is required for mammary gland development. Analysis of TCGA data reveals that somatic heterozygous mutations in GATA3 occur in up to 15% of estrogen receptor positive breast tumors, and that these tumors are diagnosed a median of eight years earlier than other estrogen receptor positive tumors, suggesting a more aggressive phenotype. These mutants have been proposed to be null alleles resulting in haploinsufficiency, however the mutation spectrum of GATA3 in breast cancer is in sharp contrast to that found in HDR syndrome, a true GATA3 haploinsufficiency disease. Based on this disparity, we propose that there is a selective pressure to mutate and retain a portion of the GATA3 in breast cancer. Here we focus on the GATA3 mutants which lack the second zinc finger which is responsible for GATA motif binding. Expression of these mutants accelerated xenograft tumor growth by ZR751 cells, and transgenic expression in mouse mammary glands promoted precocious lobuloalveolar development. We have used integrated gene expression and ChIP-Seq profiling to demonstrate that these zinc-finger deleted proteins retain the ability to associate with the genome by tethering to complexes associated with FOXA1 and AP-2gamma recognition motifs, where they modulate the expression of adjacent genes. These data support a model in which the GATA3 mutations recently observed in breast cancer encode for active transcription factors which elicit proliferative phenotypes in normal mammary epithelium and promote the growth of estrogen receptor positive breast cancer cell lines.

Citation Format: Natasha Chandiramani, Esther Peterson, Paraic A. Kenny. Mutant GATA3 actively promotes the growth of normal and malignant mammary cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1991.
Abstract 1992: Exploring target genes of eukaryotic initiation factor 5A in cancer using RNA sequence analysis
Authors: Muramatsu Tomoki , Tanimoto Kousuke , Inazawa Johji
Published: July 2016

Protein synthesis has a key role of maintaining cellular homeostasis such as growth and metabolism. However, dysregulation of translational process is a frequent feature of cancer and contributes to cancer progression. Since oncogenes and tumor suppressor genes are regulated by the translation machinery, therapeutic drugs which target the translation machinery have the possibility for overcoming cancer. We then focused on the hypusine cascade which is related to translational process. In our previous study, we showed that overexpression of the deoxyhypusine synthase (DHPS), an enzyme contained in the hypusine cascade, promoted cancer progression such as migration and invasion ability through RhoA signaling pathway. A DHPS inhibitor, GC7, showed significant decrease in the tumor formation in vivo. In particular, eukaryoutec initiation factor 5A (eIF5A) which is activated by hypusination has important roles of the translational regulation such as initiation and elongation of protein synthesis and nucleocytoplasmic transport of mRNA. We tried to identify the target genes of eIF5A by combining RNA-ChIP method with RNA sequencing analysis. As a result, candidate genes which bound to eIF5A were extracted by bioinformatics analysis, and redundant binding motifs for eIF5A in mRNAs emerged. These findings and future analyses would reveal the regulation of translational machinery by eIF5A which could be an important target for cancer therapy.

Citation Format: Tomoki Muramatsu, Kousuke Tanimoto, Johji Inazawa. Exploring target genes of eukaryotic initiation factor 5A in cancer using RNA sequence analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1992.
Abstract 1994: Post-translational regulation of the cleaved fragment of Par-4 in ovarian and endometrial cancer cells
Authors: Brasseur Kevin , Adam Pascal , Tardif Laurence , Fabi François , Parent Sophie , Asselin Éric
Published: July 2016

Prostate Apoptosis Response-4 (Par-4) is a tumor suppressor protein whose expression level in cancer is frequently decreased; however, it is rarely mutated nor suppressed. Par-4 is not only regulated at the expression level, but also post-translationally. Most recently, we reported the cisplatin-induced caspase3-dependent cleavage of Par-4 resulting in the accumulation of a 25kd cleaved-Par-4 (cl-Par-4) fragment. In the present study, we investigated the mechanisms regulating this fragment using cl-Par-4-expressing stable clones derived from ovarian and endometrial cancer cell lines.

Briefly, we used cancer cell lines in which we produced stable clones expressing the cleaved form of PAR-4 (cl.PAR-4) using lentiviral particles. Using these models, we studied the regulation of cl.PAR-4 at the protein level using Western blots.

Intriguingly, cl-Par-4 protein was weakly expressed in all stable clones despite constitutive transgene expression. However, upon cisplatin treatment, cl-Par-4 levels increased up to 50-fold relative to baseline conditions. We assessed the subcellular localization of cl-Par-4 in baseline and cisplatin-treated conditions using cytoplasmic/nuclear fractionation. Results showed that cl-Par-4 is localized in both the cytoplasm and nucleus compartments, but cisplatin treatment did not alter its localization. Treatment of stable clones with proteasome and translation inhibitors revealed that cisplatin exposure may in fact protect cl-Par-4 from proteasome-dependent degradation. To confirm proteasome involvement in this process, potential ubiquitination sites were predicted using bioinformatic tools. Interestingly, we showed that PI3K and MAPK pathways are also implicated in this post-translational regulation, as evidenced by an increase of cl-Par-4 in the presence of PI3K inhibitors (Wortmannin and NVP-BEZ235) and a decrease of cl-Par-4 using MAPK inhibitors (U0126 and PD98059). Finally using bioinformatics resources, we found diverse datasets showing similar results to those we observed with the proteasome and cl-Par-4. Indeed, we have showed, using data from a published study, that the use of Bortezomib (a potent proteasome inhibitor) significantly increased Par-4 expression in MCF7 cancer cells.

These new findings add to the complex mechanisms regulating Par-4 expression and activity, and justify further studies addressing the biological significance of this phenomenon in gynecological cancer cells.

Citation Format: Kevin Brasseur, Pascal Adam, Laurence Tardif, François Fabi, Sophie Parent, Éric Asselin. Post-translational regulation of the cleaved fragment of Par-4 in ovarian and endometrial cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1994.
Abstract 1999: Regulation of the oncogenic, invasion-promoting transcription factor myeloid zinc finger-1 (MZF1) in breast cancer by microRNAs
Authors: Kallunki Tuula , Tvingsholm Siri A. , Bundgaard Clemmensen Knut K. , Brix Ditte M. , Rafn Bo , Moreira José , Frankel Lisa , Lund Anders , Louhimo Riku , Hautaniemi Sampsa , Gromova Irina , Jäättelä Marja
Published: July 2016

INTRODUCTION: Myeloid Zinc Finger 1 (MZF1) is a transcription factor that is normally involved in myeloid differentiation. MZF1 can also be found in epithelial cancers, where it functions as an invasion and metastasis promoting oncogene. MZF1 is a central node of the invasive signaling network activated by HER2/ErbB2 in breast cancer, where MZF1 mobilizes and activates lysosomes and increases the expression of cysteine cathepsin B to promote invasion and metastasis. Thus, maintaining the sufficient expression level of MZF1 is important for the invasive signaling of breast cancer cells.

RESULTS AND EXPERIMETAL PROCHEDURES: Here we show that MZF1 is essential for the invasion of breast cancer cells in 3-dimensional cultures. We also describe a possible mechanism that regulates MZF1 levels in breast cancer. By using available micro-RNA (miRNA) binding site prediction programs (www.microrna.org/), specific microRNA mimics, corresponding miRNA inhibitors e.g. locked nucleic acids, luciferase reporter assays, immunofluorescense, quantitative PCR and 3-dimensional invasion assays we show that some breast cancer specific miRNAs can regulate MZF1 RNA and protein levels and revert the malignant and invasive cellular phenotype of breast cancer cells by altering the invasion promoting functions of lysosomes. These results are supported by the analysis of breast cancer patient data from the cancer genome atlas (http://cancergenome.nih.gov) showing that MZF1 expression correlates negatively with the expression of the identified miRNAs, when using data collected from over 600 primary breast cancer tumors.

SUMMARY: This study shows that MZF1 is an oncogene whose sufficient expression levels are central for breast cancer cell invasion and whose specific miRNA-mediated downregulation can inhibit breast cancer malignancy by modifying the expression of its lysosomal target genes.

CONCLUSION: Cancer-associated upregulation of MZF1 increases the breast cancer malignancy. Thus finding ways to downregulate MZF1 expression or inhibit its activity can be beneficial for breast cancer therapy.

Citation Format: Tuula Kallunki, Siri A. Tvingsholm, Knut K. Bundgaard Clemmensen, Ditte M. Brix, Bo Rafn, José Moreira, Lisa Frankel, Anders Lund, Riku Louhimo, Sampsa Hautaniemi, Irina Gromova, Marja Jäättelä. Regulation of the oncogenic, invasion-promoting transcription factor myeloid zinc finger-1 (MZF1) in breast cancer by microRNAs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1999.
Abstract 2005: Global binding pattern of the Wilms’ tumor gene 1 (WT1) +17AA -KTS isoform in leukemic cells
Authors: Ullmark Tove , Järvstråt Linnea , Montano Giorgia , Jernmark-Nilsson Helena , Sandén Carl , Nilsson Björn , Vidovic Karina , Gullberg Urban
Published: July 2016

The aim of this study was to investigate the global DNA-binding pattern of Wilms’ tumor gene 1 (WT1) in leukemic cells. Clinical and preclinical data indicate the zinc finger transcription factor WT1 as an oncogene, but the full target gene repertoire of WT1 in leukemic cells has not been previously characterized. The -KTS isoforms (excluding the three amino acid (KTS) insert between zinc finger three and four) are considered as the most efficient DNA-binders. Among these, the 17AA isoform (including 17 amino acids encoded by exon 5) is the most abundant one. To specifically analyze the DNA-binding of WT1(+17AA/-KTS) in leukemic cells, we generated a K562 clone that stably expressed BIO-tagged WT1(+17AA/-KTS), as well as the biotinylating enzyme Bir A. From the cells chromatin immunoprecipitation (ChIP) by streptavidin capture was performed followed by sequencing with a minimum of 50 million reads per sample. After alignment to the genome and peak calling, peaks were characterized and compared to available K562 tracks in the ENCODE database. We found that 45% of identified WT1(+17AA/-KTS) peaks are in the proximity of transcription start sites (promoter area, first exon or first intron) of target genes, whereas only 11% of randomized peaks were found here. Within the peaks we show strong enrichment for three different previously published WT1-binding motifs. Comparison to ENCODE tracks showed that WT1(+17AA/-KTS) peaks are in close proximity to binding sites of other transcription factors, to histone marks for actively transcribed genes, and to binding sites of chromatin modifiers. Considering peaks within promoters and gene bodies only (for safe assignment to a target gene), Gene Ontology (GO) analysis revealed enrichment of GO groups important for proliferation, cell death, embryonic development, and cell motility. In conclusion, WT1(+17AA/-KTS) binds close to transcription start sites in areas of active transcription. The target genes implicated in proliferation, cell death, cell signaling and motility adds to the growing evidence of WT1 as an effector gene in leukemia.

Citation Format: Tove Ullmark, Linnea Järvstråt, Giorgia Montano, Helena Jernmark-Nilsson, Carl Sandén, Björn Nilsson, Karina Vidovic, Urban Gullberg. Global binding pattern of the Wilms’ tumor gene 1 (WT1) +17AA -KTS isoform in leukemic cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2005.
Abstract 2007: Transcriptional regulatory program controlled by the oncogenic transcription factor LMO1 in neuroblastoma
Authors: Sanda Takaomi , Akahanse Koshi , Abraham Brian J. , Weichert Nina , Durbin Adam , Anders Lars , Tan Shi Hao , Yam Alice Wei Yee , Lawton Lee N. , Young Richard A. , Maris John M. , Look A Thomas
Published: July 2016

Neuroblastoma is an embryonal tumor of the peripheral sympathetic nervous system, accounting for 15% of all childhood cancer deaths. Both overexpression of the transcription factor LMO1 and the polymorphisms within this gene locus are associated with the susceptibility to neuroblastoma, but the oncogenic roles of LMO1 in neuroblastoma pathogenesis have not been elucidated. The roles of LMO1 in T-cell acute lymphoblastic leukemia (T-ALL) are better understood, and these suggest that some of its effects may be similar between the two malignancies. Here we identify the transcriptional regulatory program controlled by LMO1 in neuroblastoma and T-ALL cells. Knockdown of LMO1 induces apoptotic cell death in both tumor types. ChIP-seq and microarray analyses demonstrate that LMO1 frequently co-occupies its target genes with GATA3 and regulates gene expression in a tissue-specific manner. LMO1 positively regulates genes involved in neuronal development, tumor invasion and metastasis in neuroblastoma cells, whereas it regulates genes involved in lymphopoiesis and immune function in T-ALL cells. Gene set enrichment analysis reveals that many genes bound by LMO1 and GATA3 are significantly downregulated upon MYCN knockdown in the MYCN-amplified neuroblastoma cells. Importantly, LMO1 binds at the CDK6 gene locus, which is associated with a super-enhancer both in neuroblastoma and T-ALL cells. The mRNA expression of CDK6 is positively correlated with LMO1 expression in primary neuroblastoma samples. Knockdown of LMO1 downregulates CDK6 protein expression, whereas overexpression of LMO1 upregulates CDK6 expression. CDK6 knockdown induces apoptosis both in neuroblastoma and T-ALL cells. Our results indicate that CDK6 is a critical downstream target that is directly activated by LMO1 and is required for cell survival in neuroblastoma and T-ALL cells.

Citation Format: Takaomi Sanda, Koshi Akahanse, Brian J. Abraham, Nina Weichert, Adam Durbin, Lars Anders, Shi Hao Tan, Alice Wei Yee Yam, Lee N. Lawton, Richard A. Young, John M. Maris, A Thomas Look. Transcriptional regulatory program controlled by the oncogenic transcription factor LMO1 in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2007.
Abstract 2019: SOX2 silencing upregulates CDKN1A and suppresses growth of lung squamous cell carcinoma
Authors: Fukazawa Takuya , Yamatsuji Tomokoki , Takaoka Munenori , Yokota Etsuko , Haisa Minoru , Ishida Naomasa , Yoshida Masakazu , Miyake Noriko , Takigawa Nagio , Maeda Yutaka , Naomoto Yoshio
Published: July 2016

SOX2 is a master pluripotency controller that was recently identified as a novel major oncogene, recurrently amplified and activated in lung squamous cell carcinoma (lung SCC). Then, transcriptional downstream targets of SOX2 have been actively investigated; however, such targets are often cell line specific. Here, in order to identify highly consensus SOX2 downstream genes in lung SCC cells, we used RNA-seq data from 178 lung SCC specimens (containing tumor and tumor-associated cells) and analyzed the correlation between SOX2 and previously-reported SOX2-controlled genes in lung SCC. In addition, we used another RNA-seq dataset from 105 non-small cell lung cancer cell lines (NSCLC; including lung SCC cells) and again analyzed the correlation between SOX2 and the reported SOX2-controlled genes in the NSCLC cell lines (no tumor-associated cells). We combined the two analyses and identified genes commonly correlated with SOX2 in both datasets. Among the 99 genes reported as SOX2 downstream and/or correlated genes, we found 4 negatively-correlated (e.g., CDKN1A) and 11 positively-correlated genes with SOX2. We used biological studies to demonstrate that CDKN1A was suppressed by SOX2 in lung SCC cells. G1 cell cycle arrest induced by SOX2 siRNA was rescued by CDKN1A siRNA. These results indicate that the tumorigenic effect of SOX2 in lung SCC cells is mediated in part by suppression of CDKN1A.

Citation Format: Takuya Fukazawa, Tomokoki Yamatsuji, Munenori Takaoka, Etsuko Yokota, Minoru Haisa, Naomasa Ishida, Masakazu Yoshida, Noriko Miyake, Nagio Takigawa, Yutaka Maeda, Yoshio Naomoto. SOX2 silencing upregulates CDKN1A and suppresses growth of lung squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2019.