bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2055: miR-17-3p suppresses tumorigencity of prostate cancer through inhibiting mitochondrial antioxidant enzymes
Authors: Xu Yong , Fang Fang , Josson Sajni , Clair Daret K. St. , Clair William H. St.
Published: February 2011

Aberrant micro RNA (miRNA) expression has been implicated in pathogenesis of cancer. Recent studies have shown that miR-17-92 cluster is overexpressed in many types of cancer (Venturini et al Blood 2007; Mendell Cell 2008). The oncogenic function of mature miRNAs encoded by the miR17-92 cluster was identified from the 5’ arm of six precursor including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92-1. However, function of the miRNAs produced from the 3’ arm of these precursors remains to be elucidated. The present study demonstrates that miR-17-3p is able to suppress multiple primary mitochondrial antioxidant enzymes including manganese superoxide dismutase (MnSOD or SOD2), glutathione peroxidase-2 (GPX2), and thioredoxin reductase-2 (TXNRD2). Transfection of miR17-3p into prostate cancer PC-3 cells significantly reduced levels of the three antioxidant proteins and activity of the luciferase reporter under the control of identified miR-17-3p blinding sequences located in the 3’-untranslated regions of the three target genes. Disulfiram, a drug used to treat chronic alcoholism and also shown to have anticancer effect, induces the level of mature miR-17-3p and apoptotic cell death in PC-3 cells, which can be attenuated by transfection of antisense miR-17-3p. Increasing miR17-3p level in PC-3 cell by a Tet-on-based conditional expression system markedly suppressed its tumorigencity and enhanced its radiosensitivity. These results suggest that miR-17-3p may suppress aggressiveness of prostate cancer through inhibition of mitochondrial antioxidant enzymes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2055.
Abstract 2059: The role of microRNA-196b in cervical cancer
Authors: How Christine , Hui Angela B. , Fyles Anthony W. , Liu Fei-Fei
Published: February 2011

Introduction: MicroRNAs (miRs) are ∼22-nucleotide, non-coding RNAs that regulate gene expression post-transcriptionally. Recent data have indicated that miRs are aberrantly expressed in almost all human malignancies, including cervical cancer. In order to better understand how miRs might mediate tumor progression in cervical cancer, dysregulated miRs were identified and functional studies performed for miR-196b.

Experimental Procedures: miR expression was measured in 3 cervical cancer cell lines (SiHa, ME-180 and HT3) and 3 normal cervix tissues using a quantitative real-time PCR approach examining 365 miRs and 3 endogenous controls. SiHa cells were transfected with Pre-miR miRNA Precursor Molecules (Ambion) to determine the effects of miR-196b overexpression. Candidate miR-196b targets were elucidated using a tri-pronged approach combining in silico target prediction algorithms, clinical mRNA expression data, and in vitro mRNA expression data following transfection of cell lines. To test for direct binding of miR-196b to the HOXB7 3’ UTR, a luciferase reporter assay was performed. Reporter vectors were constructed containing either the HOXB7 3’ UTR sequence (pMIR-REPORT-HOXB7/UTR), or the HOXB7 3’ UTR sequence with a mutation in the predicted miR-196b binding site (pMIR-REPORT-HOXB7/mut). SiHa cells were serially transfected with: a) miR-196b pre-miR or negative control pre-miR, and b) pMIR-HOXB7/UTR or pMIR-HOXB7/mut, and luciferase activity was measured 24 hours later. To determine the effect of knocking down HOXB7, SiHa cells were transfected with siHOXB7 (Qiagen).

Results: Twenty-seven miRs were observed to be downregulated in all three cell lines compared to normal cervix. One of the 27 downregulated miRs, miR-196b, was shown to inhibit cell growth and colony-forming ability when overexpressed in SiHa cells. HOXB7 was identified as one of the candidate targets of miR-196b using our tri-pronged approach. Knocking down HOXB7 recapitulated the inhibition in cell growth and clonogenicity that was observed following mir-196b overexpression. When the luciferase reporter assay was performed, miR-196b overexpression resulted in a significant reduction in luciferase activity in cells transfected with pMIR-HOXB7/UTR, but not in cells transfected with pMIR-HOXB7/mut, demonstrating direct and specific binding of miR-196b to the 3’ UTR of HOXB7.

Conclusion: miR-196b is downregulated in cervical cancer and negatively regulates HOXB7.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2059.
Abstract 207: Distinct TRAIL resistance mechanisms can be overcome by proteosome inhibition
Authors: Menke Christina R. , Bin Lianghua , Thorburn Jacqueline , Behbakht Kian , Ford Heide , Thorburn Andrew
Published: February 2011

A major impediment to the use of Tumor Necrosis Factor-Like Apoptosis Inducing Ligand (TRAIL) Receptor targeted drugs is that many tumors evolve resistance to these agents. On the other hand however, multiple different kinds of drugs are reported to be able to synergize with TRAIL. This raises the question of whether it is possible to overcome resistance using the same drugs that cause synergy. We tested this idea by creating a series of isogenic tumor cells with acquired TRAIL resistance or defined mechanisms of resistance that are known to occur in human tumors then compared these cells to the TRAIL-sensitive parental cell line. Although diverse classes of anti-cancer drug were all able to synergize with TRAIL most of these agents were unable to overcome resistance and there was no correlation between the amount of synergy that is seen with a particular agent and its ability to overcome acquired resistance. Proteosome inhibitors were however able to overcome diverse resistance mechanisms suggesting that one should select drugs for TRAIL R agonist combination therapy based not just on their ability to synergize but rather on their ability to both overcome resistance and synergy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 207.
Abstract 2070: Mir-199a is downregulated in hypoxia and targets HIF1α in ovarian cancer cells
Authors: Joshi Hemant , Subramanian Indira , Ghosh Goutam , Zeng Yan , Zhao Min , Ramakrishnan Sundaram
Published: February 2011

HIF1 is the main transcriptional regulator of the hypoxic response in mammalian cells and plays an important role in the adaptation of tumor cells to hypoxic microenvironments. While normally degraded under normoxic conditions via oxygen-dependent, ubiquitin-based mechanisms, HIF1α protein, which, with the constitutively expressed HIF1β forms the HIF1 heterodimer, is stabilized in hypoxic tumor cells. Stabilization of HIF1α protein under hypoxia leads to transactivation of a variety of angiogenic and tumorigenic genes including VEGF and matrix metalloproteinase 2. Clinically, high levels of HIF1 in tumors translate to increased patient morbidity and mortality. Our studies show that HIF1α, in addition to being regulated at the protein level, is also directly regulated by microRNAs, small 20-22 base ribonucleotides that specifically bind to the 3’ untranslated regions (UTRs) of target mRNA to inhibit translation or induce degradation. Based on bioinformatics and microRNA-target databases, we determined that HIF1α contains a potential binding site in its 3’ UTR for mir-199a, which, based on our microarray and RT-qPCR data, is significantly downregulated in hypoxic ovarian cancer cells compared to normoxia. Using a luciferase reporter construct in which luciferase expression is under control of the HIF1α 3’ UTR, we showed that overexpression of mir-199a in A2780 or MA148 cells leads to suppression of the luciferase signal. Mutation of the mir-199a binding site in the HIF1α 3’ UTR resulted in no change in luciferase signal with mir-199a overexpression, indicating that mir-199a directly targets the HIF1α 3’ UTR. Furthermore, decreasing mir-199a levels, either by incubating cells in hypoxia or using a morpholino specifically targeting mir-199a, leads to increased levels of HIF1α protein as determined by western blot and increased transcripts of HIF1-activated genes such as VEGF as measured by RT-qPCR. Conversely, overexpression of mir-199a leads to decreased HIF1α protein and VEGF transcript levels. Together, our findings have significant implications in the study of HIF1α regulation in cancers. Mir-199a may have a role in future therapeutic strategies to modulate the tumor microenvironment to limit hypoxia-driven tumor progression and metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2070.
Abstract 2075: MicroRNA-21 expression levels are accompanied by respective alterations in pdcd4 protein levels in non-small cell lung cancer
Authors: Sourvinou Ioanna S. , Markou Athina , Kaklamanis Loukas , Tsaroucha Emily G. , Ozaki Iwata , Matsuhashi Sachiko , Lianidou Evi S.
Published: February 2011

Introduction: MicroRNA-21 is a well characterized cancer associated miRNA as it has been found overexpressed in nine types of solid tumors and in other malignancies. The role of miR-21 is related with diverse carcinogenesis processes. The downregulation of tumor suppressor genes through the overexpression of miR-21 has been demonstrated for PTEN and TPM1. Very recently, it was revealed that programmed cell death 4 (PDCD4) is another target of miR-21 in colon and breast cancer cell lines. As it has not been yet studied the correlation between miR-21 and PDCD4 levels in clinical samples, we conducted the present study to investigate the possible regulation of pdcd4 protein levels by mature miR-21.

Materials and Methods: Forty pairs of NSCLC fresh-frozen tissues and their corresponding noncancerous tissues were analyzed for the expression of mature miR-21 using quantitative real-time RT-PCR, as previously described (Markou et al., 2008). In parallel, PDCD4 protein levels were evaluated by immunohistochemistry. Deparaffinized sections cut from paraffin-embedded tissue samples were stained with a specific anti-PDCD4 antibody (1:100 dilution) (Ozaki et al., 2006) using HRP DAB kit (DAKO) for the detection. The tumor types and stages were determined according to the WHO classification. All samples were analyzed histologically to access the amount of tumor component (at least 70% of tumor cells) and the quality of material.

Results: Among the 40 NSCLC tissue specimens studied, suppresion of miR-21, in respect to their adjacent non-neoplastic tissues, was detected in 24 samples (60 %). In 15 out of these 24 patients (62.5%), we observed that the supression of miR-21 was accompanied by increase of PDCD4 protein levels. Mature miR-21 was overexpressed in 16 out of 40 patients (40%), and in 8 out of these 16 patients (50%) we detected reduced PDCD4 protein levels. Totally, in 23 out of 40 samples (57.5%), the altered miR-21 expression levels correlated with changed PDCD4 protein levels.

Conclusion: Our data indicate for the first time that PDCD4 protein expression levels are regulated by miR-21 in non small cell lung cancer tissues.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2075.
Abstract 2080: miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma
Authors: Furuta Mayuko , Kozaki Ken-ichi , Tanaka Shinji , Arii Shigeki , Imoto Issei , Inazawa Johji
Published: February 2011

MicroRNAs (miRNAs) are a class of small non-coding RNAs that, in general, negatively regulate gene expression. They have been identified in various tumor types, showing that different sets of miRNAs are usually deregulated in different cancers. Some miRNA genes harboring CpG-islands undergo methylation-mediated silencing, a characteristic of many tumor-suppressor genes. To identify such miRNAs in hepatocellular carcinoma (HCC), we first examined the methylation status of 43 loci containing CpG-islands around 39 mature miRNA genes in a panel of HCC cell lines and non-cancerous liver tissues as controls. Among 11 miRNA genes frequently methylated in HCC cell lines but not in non-cancerous liver tissues, 3 miRNA genes, i.e. miR-124, miR-203, and miR-375, were selected as silenced miRNAs through CpG-island methylation by comparing methylation and expression status and evaluating restored expression after treatment with 5-aza-2′-deoxycytidine. In primary tumors of HCC with paired non-tumorous liver tissues, only miR-124 and miR-203 showed frequent tumor-specific methylation, and their expression status was inversely correlated with methylation status. Ectopic expression of miR-124 or miR-203 in HCC cells lacking their expression inhibited cell growth, with direct down-regulation of possible targets, cyclin-dependent kinase 6 (CDK6), vimentin (VIM), SET and MYND domain containing 3 (SMYD3), and IQ motif containing GTPase activating protein 1 (IQGAP1) or ATP-binding cassette, sub-family E, member 1 (ABCE1), respectively. Our results suggest that miR-124 and miR-203 are novel tumor-suppressive miRNAs for HCC epigenetically silenced and activating multiple targets during hepatocarcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2080.
Abstract 2089: MiR-205 role in triple negative breast cancer
Authors: Iorio Marilena V. , Piovan Claudia , Leva Gianpiero Di , Casalini Patrizia , Tortoreto Monica , Plantamura Ilaria , Taccioli Cristian , Croce Carlo M. , Tagliabue Elda
Published: February 2011

An increasing number of experimental evidences show that microRNAs can have a causal role in breast cancer tumorigenesis as a novel class of oncogenes or tumor suppressor genes, depending on the targets they regulate. miR-205, down-modulated in breast cancer versus normal breast tissue, was originally associated to absence of vascular invasion and shown to be involved in the EMT. Moreover, we recently demonstrated that miR-205 directly targets HER3 receptor and impairs the Akt-mediated survival pathway, thus inhibiting clonogenic potential of SKBr3 cells and increasing the responsiveness to tyrosin-kinase inhibitors.

Considering the striking evidences of a major role of miR-205 as oncosuppressor in breast cancer, we decided to investigate its possible involvement in another tumor subgroup, extremely aggressive and still lacking a specific therapy: triple negative breast tumors. Here we show that re-introduction of miR-205 in the triple negative breast cancer cell line MDA-MB-231 led to a dramatic reduction of both proliferation and clonogenic potential as well as migration capability. Notably, miR-205 also significantly inhibited the in vivo growth in xenograft models obtained injecting stable clones of MDA-MB-231 cells transfected with a miR-205-encoding plasmid or the empty vector.

Investigating alternative pathways potentially regulated by miR-205 in this model, we noticed that many of the predicted targets are actually interconnected and related to adhesion and migration processes. Indeed, this microRNA seems to be involved in the control of cell-cell and cell-matrix adhesion regulating the expression of extracellular matrix-related molecules. Among them, we focused on LAMC1, reported to be involved in cell adhesion, differentiation, migration, signaling and metastasis. By Western Blot and Luciferase assay, we demonstrated that miR-205 directly targets LAMC1 in MDA-MB-231 cells.

In conclusion, miR-205 exerts a role as oncosuppressor gene also in triple negative breast cancer: it exerts a dramatic inhibition of proliferation and migration in triple negative MDA-MB-231 cell line, at least partially through direct regulation of LAMC1. These evidences not only underline the consistent role of miR-205 in the biology of different breast cancer subgroups, but provide the rational bases for a therapeutic intervention miRs-based.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2089.
Abstract 2091: MiR-145 functions as a potential tumor suppressor in hepatocellular carcinoma
Authors: LAW Priscilla TY , Ching Arthur KK , Wong Queenie WL , Lai Paul BS , Wong Nathalie
Published: February 2011

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. The pathogenesis of HCC has been associated with various genetic and epigenetic alterations, including microRNA dysregulation. In defining the differentially expressed miRNAs in HCC, we previously reported on HCC-specific microRNA deregulations by array-based profiling, which revealed common down-regulations of miR-145. Subsequent verification of miR-145 expression in an independent cohort of 72 HCC cases by qRT-PCR corroborated significant down-regulations in tumors compared to their non-malignant counterparts (49%; paired t-test p=0.026). Transfection of miR-145 into HCC cell lines, HKCI-C2 and HKCI-9, showed a consistent inhibitory effect on both cell viability and proliferation as indicated by MTT and colony formation assays. Flow cytometric analysis on HKCI-C2 further indicated that miR-145 could induce G2/M cell cycle arrest and increase apoptosis. MiR-145 has been shown to target insulin receptor substrate 1 (IRS1) in colorectal cancer. It is known that IRS1 is a substrate of insulin-like growth factor receptor (IGF-1R) in the IGF signaling pathway. In-silico prediction by Targetscan, miRNAda and RNAhybrid suggested that miR-145 might also target IGF-1R and IRS2 along the IGF axis. The effects of miR-145 on these IGF components were hence investigated. Our results from Western blot indicated that IRS1 expression was down-regulated in the presence of miR-145, suggesting that it might also be a target of miR-145 in HCC. Taken together, we found that miR-145 could induce cell cycle arrest and apoptosis in HCC cells, possibly through its effect on IRS1. This study reveals for the first time that miR-145 may act as a tumor suppressor in the liver tumorigenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2091.
Abstract 2093: Functional screening of tumor-suppressive microRNAs silenced by DNA hypermethylation in oral squamous cell carcinoma
Authors: Kozaki Ken-ichi , Uesugi Atsushi , Furuta Mayuko , Imoto Issei , Inazawa Johji
Published: February 2011

In human cancer, the DNA hypermethylation of CpG-sites located within CpG islands is one of important molecular mechanisms causing the down-regulation of microRNAs (miRNAs) in a similar manner to that demonstrated for classical tumor-suppressor genes. In this study, to identify tumor-suppressive miRNAs (TS-miRs) silenced by hypermethylation during oral carcinogenesis, we first performed function-based screening for TS-miRs in 327 miRNA in 2 oral squamous cell carcinoma (OSCC) cell lines with Pre-miR™ miRNA Precursor Library-Human V2. Remarkable growth inhibition was observed in 110 miRNAs (33.6%) as compared with precursor negative control, and 61 miRNAs of these miRNAs were localized around CpG-islands. Among these 61 miRNAs, we further analyzed DNA hypermethylation by combined bisulfite restriction analysis (COBRA) in 33 loci containing CpG-islands around 33 mature miRNAs, except 28 miRNAs which have been already reported as TS-miRs or targets for DNA methylation for silencing in cancer cells, resulting in identification of some miRNAs as the candidate TS-miRs silencing by CpG island hypermethylation. Expression levels of these candidate miRNAs were inversely correlated with their DNA methylation status, and were restored by treatment with 5-aza 2’-deoxycytidine in OSCC cells. Moreover, the ectopic transfection of these miRNAs into OSCC lines lacking their expressions significantly reduced cell growth. Taken together, our results clearly demonstrate that these candidates are TS-miRs epigenetically silenced during oral carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2093.
Abstract 2095: The functional significance of microRNA-145 in prostate cancer
Authors: Zaman Mohd S. , Deng Guoren , Shahryari Varahram , Suh Seong O. , Chen Yi , Tanaka Yuichiro , Dahiya Rajvir
Published: February 2011

MicroRNAs are small noncoding RNAs that play important roles in numerous cellular processes including development, proliferation, and apoptosis. Recent studies have demonstrated aberrant expression of miRNAs in prostate cancer tissues and cell lines. miRNAs microarray data, showed that miR-145 is down-regulated in prostate cancer. Therefore, we investigated the expression and functional significance of miR-145 in prostate cancer. The expression of miR-145 was found to be low in all of the prostate cell lines tested (PC3, LNCaP and DU145). The level of miR-145 expression in adjacent normal regions was found to be significantly higher as compared to the matched cancerous regions. Over expression of miR-145 in PC3 transfected cells resulted in a significant increase in apoptosis and an increase in cells in the G2/M phase, as detected by flow cytometry. Our results also show that methylation of the promoter region of miR-145 in cell lines and carcinoma samples as one of the reasons for its low expression. To identify potential targets for miR-145, we performed microarray analyses in miR-145 over-expressing PC3 cells and observed up regulation of the pro apoptotic gene TNFsf10 which was confirmed by real-time PCR and Western analysis. This is the first report to show that miR-145 acts as a tumor suppressor in prostate cancer through cell cycle arrest and induction of apoptosis. Since a major target for miR-145 is the pro apoptotic TNFsf10 gene, modulation of miR-145 may provide a therapeutic approach for the management of prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2095.
Abstract 2096: The role of microRNA-1 as a candidate tumor suppressor in cutaneous squamous cell carcinoma
Authors: Gable Dustin L. , Fleming Jessica L. , Toland Amanda Ewart
Published: February 2011

Cutaneous squamous cell carcinoma (SCC) is the second most common form of skin cancer, affecting over 250,000 people in the United States annually. Using miRNA expression arrays, we identified lower levels of microRNA-1 (miR-1) expression in skin from FVB/NJ mice, which are susceptible to chemically-induced skin cancer, compared to skin from Spr/EiJ mice, which are resistant to chemically-induced skin cancer. Additionally, murine SCC cell lines showed greatly reduced levels of miR-1 compared to normal skin. We hypothesize that decreased expression of miR-1 in susceptible mice and SCC cell lines suggests a possible role of miR-1 as a tumor suppressor in skin. To test this hypothesis, we transfected a precursor for miR-1 into a murine SCC cell line, A5, and measured proliferation and cell viability via an MTT assay at 24, 48, and 72 hours post-transfection. The growth rate for cells transfected with miR-1 was significantly decreased at 48 to 72 hours post-transfection relative to those mock-transfected cells and corresponded to miR-1 expression levels. These data are consistent with our hypothesis that miR-1 may act as a tumor suppressor in the skin. Using in silico prediction programs, we identified a number of putative targets of miR-1. The transcription factor oncogene, Ets1, emerged as an interesting potential direct target of miR-1. Ets1 has three predicted target binding sites for miR-1. Quantitative PCR and Western analyses revealed decreased Ets1 mRNA and protein expression in A5 cells transfected with miR-1 compared to mock-transfected A5 control cells. Ets1 mRNA expression is also decreased in a normal keratinocyte cell line, C5N, compared to A5 cells. From these experiments we showed that Ets1 expression shows an inverse correlation with miR-1 levels. These results suggest Ets1 is regulated by miR-1. Future studies will determine whether Ets1 is a direct target of miR-1. We will also clarify the role of Ets1 in SCC cell proliferation and migration and identify other tumor-related phenotypes associated with miR-1.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2096.
Abstract 2100: Down regulation of HOXD10 by microRNA-10b enhances proliferation of glioblastoma multiforme derived cell lines
Authors: Gaur Arti B. , Israel Mark A.
Published: February 2011

We sought to elucidate the role of a potentially oncogenic microRNA, microRNA-10b (mir-10b), in mediating the pathology of glioblastoma multiforme (GBM). MicroRNAs are small noncoding RNAs that play a critical role in developmental and physiological processes and are implicated in the pathogenesis of several human diseases including cancer. MicroRNAs function by regulating target gene expression post-transcriptionally. We have previously assessed the expression pattern of 241 mature human microRNAs in the NCI60 panel of cell lines and in a set of corresponding normal tissues (Gaur et al., 2007, Cancer Research. 67(6):2456-68). We found that microRNA expression patterns mark specific biological characteristics of tumors and/or mediate biological activities important for their pathobiology. In CNS tumor-derived cell lines, we identified mir-10b as a candidate oncogene, as it is expressed at higher levels in human GBM lines an primary GBM tissue than it is in normal brain tissue. Using several target prediction algorithms we identified a member of the homeobox family, Homeobox D10 (HOXD10), as a potential target of mir-10b. Our in vitro studies demonstrate that down regulation of mir-10b in GBM-derived cell lines results in increased expression levels of HOXD10. Additionally, our data indicate that down-regulation of mir-10b in human GBM lines leads to decreased proliferation and decreased anchorage independent colony formation in soft agar. Importantly, using a xenograft model in immune-deficient nude mice, we have observed human that GBM lines in which mir-10b expression is inhibited exhibit decreased tumor formation and growth in vivo. Furthermore, the decreased colony formation in soft agar observed as a result of mir-10b inhibition in GBM-derived cell lines is significantly antagonized by expression of siRNAs complementary to HOXD10 mRNA. Also, over expression of HOXD10 in GBM cell lines results in differentiation and decreased proliferation. Further experiments are ongoing to identify the specific role of HOXD10 in mediating the pathologic characteristics of GBM. An understanding of the pathways mediated by HOXD10 signaling may provide novel opportunities for therapeutic intervention. The National Brain Tumor Society (ABG) and the Theodora B. Betz Foundation (MAI) supported this work.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2100.
Abstract 2101: GBM: miR-34a acts as a tumor suppressor and prognosis maker as well as modulating EGFR
Authors: Yin Dong , Ogawa Seishi , Kawamata Norihiko , Thoennissen Nils H. , Chen Weikai , Leiter Amanda , Ham Michelle , Doan Ngan B B. , Said Jonathan W. , Black Keith L. , Koeffler H. Phillip
Published: February 2011

The whole genomic gene copy number was measured using single nucleotide polymorphism DNA microarray (SNP-Chip). Chromosome 1p36.23 was frequently deleted (19 of 55 clinical samples, 35 %) and EGFR was often amplified (35 of 55, clinical samples 66 %) in glioblastoma multiform (GBM). microRNA 34a (miR-34a) localizes in this minimum common deleted 1p36.23 region. We hypothesized that miR-34a may act as a tumor suppressor in the brain. Interestingly, we found that the mean survival time was significantly shorten (p < 0.05) in the patients whose GBM showed both EGFR amplification and miR-34a deletion compared to those with either EGFR amplification, miR-34a deletion or normal levels of EGFR and miR-34a. Expression of miR-34a was significantly lower in GBM samples compared to normal brain tissue as examined using real-time RT PCR. Forced expression of miR-34a in GBM cell lines decreased their cell growth both in liquid culture and in immunodeficient mice; and the latter was associated with decreased angiogenesis. These cells also had decreased ability to migrate as detected by Boyden chamber and had profoundly low levels of cell cycle proteins (Cyclin -A1, -D1, -D3 and -B1, CDK2, as well as, E2F1, −3, and −4) and increased expression of cyclin kinase inhibitor (CKI) proteins, p21 and p27. Furthermore, the protein expression of EGFR was decreased in the cells with overexpression of miR-34a. Taken together, GBMs often acquire a miR-34a deletion and have low expression of this microRNA. The miR-34a probably normally acts as a tumor suppressor by inhibiting growth of GBM cells in vitro and in vivo associated with moderating the expression of cell cycle proteins and EGFR. Individuals whose GBM have both deletion of miR-34a and amplification of EGFR have a particularly poor prognosis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2101.
Abstract 2105: miR-125b promotes the tumor growth of prostate cancer cells in intact and castrated male nude mice
Authors: Shi Xu-Bao , Xue Lingru , deVere White Ralph W.
Published: February 2011

Non-coding microRNAs (miRNAs) negatively regulate protein-coding genes, and aberrant expression of certain miRNAs has been reported to play a key role in the pathogenesis of human cancer including prostate cancer. Our previous studies have shown that miR-125b was highly expressed in a majority of clinical prostate cancer tissues we examined and ectopic expression of miR-125b stimulated the in vitro growth of LNCaP cells in the absence of androgens. In this study, we performed in vivo experiments to further determine whether this miRNA promotes prostatic xenograft growth. To this end, a miR-125b lentiviral vector (lenti-miR-125b) that expresses a ∼500-base pre-miR-125b was used to infect androgen-dependent PC-346C prostate cancer cells that have a limited ability to form tumors in intact nude mice and fail to grow in castrated nude mice. Quantitative RT-PCR exhibited a 39-fold increase in miR-125b level in lenti-miR-125b infected cells compared to that in lenti-vector control cells. The effect of miR-125b on xenograft tumor growth was then evaluated in intact and castrated male nude mice. After inoculation with lenti-miR-125b cells or control cells, tumors formed in all intact mice. However, the palpable lenti-miR-125b tumors appeared in approximately 7 days, while the vector control tumors took 15-20 days. The volume of xenograft tumor was markedly enlarged in all lenti-miR-125b mice compared to that in control mice. Surgery castration was performed when the tumor volume reached ∼0.5 cm3. Castratation resulted in temporary growth regression of miR-125b tumors followed by rapid growth of these tumors. To understand the molecular mechanism by which miR-125b promotes tumor growth in intact and castrated nude mice, xenograft tumors were examined by Western blot analyses of the expression of p53, Puma and Bak1 that have been validated to be the direct targets of miR-125b. Marked reduction of these three pro-apoptotic molecules was observed in miR-125b tumors compared to that in control tumors. Our data indicate that miR-125b acts as an oncogenic molecule, and overexpression of miR-125b stimulates the in vivo growth of prostate cancer cells as well as promotes the progression of prostate cancer cells to an androgen-independent status.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2105.
Abstract 2107: Deregulation of tumor suppressor miRNAs results in Six1 oncogene overexpression in pediatric and adult human cancers
Authors: Imam J. Saadi , Buddavarapu Kalyan , Lee-Chang Jennifer S. , Hornsby Peter , Chen Yidong , Yoon Kihoon , Rao Manjeet
Published: February 2011

MicroRNAs (miRNAs) are naturally occurring, short non-coding RNAs believed to be involved in many biological processes including normal development and adult cancer. It seems logical that miRNAs would be particularly important in pediatric cancers as these malignancies generally involve perturbations of normal developmental and differentiation processes, yet relatively little is known about the role of miRNAs in pediatric cancers. Here, we show that oncogenesis in Wilms’ tumor - the most common pediatric renal cancer - is mediated by miRNAs. MiRNA microarray analysis identified a cohort of miRNAs that show altered expression in Wilms’ tumors when compared with normal matched control kidney. Our results reveal that several of these miRNAs show similar expression patterns both in Wilms’ tumors and fetal kidney, suggesting that they may play an important regulatory role in kidney development and Wilms’ tumorigenesis. Supporting this notion are our findings that show a number of differentially expressed miRNAs to target genes known to play a role in kidney development and Wilms’ tumorigenesis, including the Six1 homeobox gene, a potent oncogene reported to be overexpressed in several solid tumors. Analyses of primary Wilms’ tumors and ovarian cancer tissues as well as multiple tumor cell lines from breast cancer, ovarian cancer and rhabdomyosarcoma showed decreased expression of miRNAs targeting the Six1 3′UTR, paralleling an increase in Six1 levels. Further investigation revealed a potent tumor suppressor role for Six1 targeting miRNAs, as ectopic expression suppressed the ability of cancer cell lines to form colonies and tumor growth in nude mice. Interestingly, the reduced expression of these miRNAs in Wilms’ tumors was due in part to genomic loss of loci that contain these miRNAs, as revealed by high resolution comparative genomic hybridization analysis. In summary, our findings suggest that miRNAs are integral to the molecular circuitry that plays an important role in Wilms’ tumorigenesis by regulating events during normal kidney development. In addition, our discovery of novel tumor suppressor miRNAs targeting the Six1 oncogene promises to unravel a unique regulatory mechanism for Six1 deregulation in pediatric as well adult tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2107.
Abstract 2109: Mir-125b confers the resistance of cancer cells to Taxol through suppression of Bak1
Authors: Zhou Ming , Zhao Yuhua , Ding Yan , Liu Hao , Liu Zixing , Xi Yaguang , Xiong Wei , Li Guiyuan , Lu Jianrong , Fodstad Oystein , Riker Adam I. , Tan Ming
Published: February 2011

Paclitaxel (Taxol) is one of the most effective chemotherapeutic agents for treatment of cancer patients. Despite impressive initial clinical responses, the majority of patients will eventually develop some degree of resistance to Taxol-based therapy. The mechanisms underlying cancer cells resistance to Taxol are not fully understood. microRNA (miRNA) has emerged to play important roles in tumorigenesis and drug-resistance. However, the interaction between the development of Taxol resistance and miRNA has not been previously explored. In this study, we utilized a miRNA array to screen differentially expressed miRNAs, comparing Taxol-resistant and their Taxol-sensitive parental cells, and investigated the role and mechanisms of miR-125b in Taxol resistance of breast cancer cells. We found that miR-125b was up-regulated in Taxol-resistant cells, with its overexpression causing a marked inhibition of Taxol-induced cytotoxicity and apoptosis, and a subsequent increase in the resistance of cancer cells to Taxol. Moreover, we demonstrated that Bak1 is a direct target of miR-125b in breast cancer cells, and down- regulation of Bak1 suppressed Taxol- induced apoptosis and led to an increased resistance to Taxol. Furthermore, restoring the expression of Bak1 by either miR-125b inhibitor or specific siRNA recovered Taxol sensitivity, overcoming miR-125-mediated Taxol resistance. Taken together, our data strongly supports a central role for miR-125b in conferring Taxol resistance through the suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming Taxol resistance in a number of different tumor histologies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2109.
Abstract 2112: COLD-PCR -based deep sequencing identifies novel mutations and intratumor TP53 mutational heterogeneity in lung adenocarcinoma
Authors: Li Jin , Milbury Coren A. , Li Cheng , Makrigiorgos Mike G.
Published: February 2011

TP53 is frequently mutated in lung adenocarcinoma, a major type of lung cancer, and certain TP53 mutations correlate with prognosis and multidrug resistance. Reliable identification of the type and position of TP53 mutations is problematic as the mutations are scattered over numerous codons while their abundance in clinical samples can fall below the sensitivity limits of conventional sequencing methods. Here we employ COLD-PCR, a new form of PCR that selectively amplifies mutation-containing sequences such that the resulting PCR products contain high percentages of mutant alleles. To ensure the highest sensitivity, we adapted the method to employ two consecutive rounds of COLD-PCR followed by Sanger sequencing. Using this highly sensitive new approach for ‘deep-sequencing’ we screened 48 lung adenocarcinoma samples to identify presence and type/position of low-level TP53 mutations. Missense/frame-shift TP53 mutations throughout exons 5-8 were identified in 23 out of 48 (48%) lung adenocarcinoma samples examined, including 8 low-level mutations with an abundance of 1-17% and 3 with an abundance below 1%. 3 novel lung adenocarcinoma mutations were discovered. We identified disruptive mutations that are undocumented in the lung cancer TP53 mutation database and samples harboring low-level (2%-abundance) putative driver mutations concurrently with passenger clonal TP53 mutations (80%-abundance). Our data reveal the presence of widespread intra-tumoral TP53 mutation heterogeneity, and a spectrum of numerous protein-altering mutations at low abundance, that were missed in previous investigations. The implications of using deep-TP53-sequencing as a prognostic/predictive biomarker, evaluation of cancer risk, recurrence, and further understanding of cancer biology are discussed. In addition, we evaluate the use of whole genome-amplification prior to mutation screening in situations when the source of genomic DNA is limited, which can be critical for early cancer detection from biopsy, the assessment of minimal residual disease following surgery, disease staging, and monitoring of cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2112.
Abstract 2114: Deletions/Duplications detection by MLPA in Chilean families with hereditary colorectal cancer: Lynch syndrome and FAP
Authors: Hurtado Claudia , Orellana Paulina , Alvarez Karin , De la Fuente Marjorie , Wieland Ana María , Pinto Eliana , Heine Claudio , Church James , Kronberg Udo , Carvallo Pilar , López-Kostner Francisco
Published: February 2011

The main variants of colorectal cancer hereditary are Familial Adenomatous Polyposis (FAP) and Lynch Syndrome, which account for approximately 5% of total cases of colorectal cancer. Point mutations in APC and MMR genes are responsible of about 85% and 50% of Chilean FAP (classic phenotype) and Lynch Syndrome (Amsterdam criteria) families, respectively. Different molecular strategies are available for the detection of mutations in these genes. Studies in other populations have identified deletions and duplications of one or more consecutive exons account for around 50% and 10-20% of the total alterations in MMR and APC genes, respectively. The aim of our work was the detection of genomic deletion/duplication through Multiplex Ligation-Dependent Probe Amplification (MLPA) in Lynch Syndrome and FAP patients in Chile. In this study, we analized alterations in MLH1 and MSH2 in 26 Lynch Syndrome families (5 Amsterdam and 21 Bethesda families), and in APC in 10 FAP families (6 classic and 4 attenuated), all of them non carriers of point mutations in these genes. We identified 3 different alterations in the MLH1 in four Amsterdam families, all of them are 0.5 times deletion, which are located: exon 1 in one family, exon 19 in two families, and exons 14 and 15 in one family. In the MSH2, in one Amsterdam family we detected a 0.5 time deletion of exon 2. In the APC, we found 2 different alterations in three classic FAP families: in one family we detected a 0.5 times deletion of the whole gene including promoter region, and in two families we found a 1.5 time amplification of exons 1, 2 and 3. These alterations were confirmed in other relatives of these families and in two independent MLPA analysis. The deletion of exon 19 in MLH1 and the amplification of exons 1, 2 and 3 in APC have not been previously described in other populations. In conclusion, genomic deletions/duplications were only detected in 5 Lynch Syndrome families that fulfilled Amsterdam criteria, and in 3 FAP families with classic phenotype. The detection of different mutations involved in these syndromes, allows the development of prevention strategies of these diseases. Financed by Cleveland Clinic Foundation and Las Condes Clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2114.
Abstract 2115: Increased sensitivity of KRAS mutation detection by High-Resolution Melting analysis of COLD-PCR products
Authors: Daugaard Iben L. , Kristensen Lasse S. , Kjeldsen Tina , Dutoit Stephen Hamilton , Hager Henrik , Hansen Lise Lotte
Published: February 2011

Much effort has been put into the development of sophisticated technologies enabling the detection of clinically significant low-level tumor specific KRAS mutations. The constitutively active KRAS oncogene harbors a mutation hotspot in codon 12 and 13 of exon 2. These mutations are predictors of response to treatment with the monoclonal antibodies panitumumab and cetuximab targeting the epidermal growth factor receptor (EGFR). Therefore, it is crucial to develop robust and sensitive assays targeting these mutations. Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that selectively amplifies mutation containing amplicons based on the lower melting temperature of heteroduplexes versus homoduplexes. We have developed an assay based on real-time COLD-PCR and high-resolution melting (HRM) targeting codon 12 and 13 for simultaneous detection of the various mutations found in this mutation hotspot. By using mutation containing cell-lines serially diluted into wild-type DNA we have compared the sensitivity of HRM after standard PCR with the sensitivity of HRM after COLD-PCR. We have also analyzed 60 colorectal cancer samples for which KRAS mutation status previously has been evaluated by the DxS kit which is approved by the U.S. Food and Drug Administration. Replacing standard PCR by COLD-PCR increased the sensitivity of the assay by up to 50 fold depending on the specific mutation. Furthermore, we have obtained very similar results when using our COLD-PCR assay as we obtained when using the DxS kit and confirmed the HRM results with Sanger sequencing. In conclusion, applying COLD-PCR to HRM analysis is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2115.