bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3003: Immune cell-related microRNA-155 is associated with human liver cirrhosis and hepatocellular carcinoma
Authors: Budhu Anuradha S. , Yu Zhipeng , Forgues Marshonna , Tang Zhao You , Croce Carlo M. , Wang Xin Wei
Published: February 2011

Liver cirrhosis, a major hepatocellular carcinoma (HCC) risk factor, is caused by chronic liver disease (CLD) of various etiologies with ambiguous underlying mechanisms. Gene profiling has illustrated that CLD etiologies differ and are associated with cytokine up-regulation. Recently, microRNAs (miRNAs), noncoding-RNA gene regulators, have been utilized as effective molecular biomarkers in many cancer types, including HCC. We investigated whether unique miRNAs are associated with CLD and HCC. We examined miRNA array profiles of 58 human liver tissue specimens with end-stage CLD (hepatitis B virus, hepatitis C virus, Wilson's disease, autoimmune hepatitis, alcoholic liver disease and primary biliary cirrhosis) and a normal liver pool. We compared the differentially expressed miRNAs between CLDs and normal liver, among CLDs and between etiologically equivalent CLD and HCC. Significant differentially expressed CLD-miRNAs were validated by quantitative-real time PCR (qRT-PCR). MiRNA and immune cell expression was measured in CLD, HCC or human peripheral blood mononuclear cells (PBMC) by qRT-PCR or immunohistochemistry. Correlation analysis between etiology-specific miRNAs and mRNAs were performed, followed by pathway analysis of the correlating mRNAs. Statistical class comparison analyses revealed that unique miRNAs were associated with each CLD group and among CLD etiologies, with only one miRNA commonly altered among CLDs (p=0.05; 10 fold cross-validation; 10k permutation). miRNA expression correlated well between array and qRT-PCR analyses (spearman r= 0.53-0.83, p= 0.03-0.001). We also found that many CLD-miRNA alterations were maintained in HCC. In particular, immune-related miR-155 was significantly up-regulated in CLDs, HCC, nontumor tissue, metastasis tissue specimens and PBMC treated with pro-metastatic colony stimulating factor 1 (CSF1) or osteopontin (OPN). MiR-155 was associated with T regulatory cells (Treg) and both were elevated by pro-inflammatory cytokines. Moreover, high miR-155 expression in nontumor tissues was associated with poor overall HCC patient survival (p=0.0244). Correlation analysis revealed that miR-155 was associated with distinct gene expression pathways in each CLD etiology. In sum, CLD miRNAs are mainly etiologically distinct and are coupled to immune responses and tumorigenesis. Our work suggests that miR-155 may modulate Tregs and hence, functionally contribute to CLD and HCC 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 3003.
Abstract 3009: Evaluating the role of cell cycle inhibition in celecoxib toxicity through microRNA analysis
Authors: Heaford Andrew C. , Erbe Christy B. , Bock Jonathan M.
Published: February 2011

Squamous cell carcinoma of the head and neck (SCCHN) is a major health problem worldwide, with over 45,000 new cases predicted to occur in the United States in 2009. Adjuvant therapies that can augment established treatments for SCCHN without causing significant additional morbidity are needed for this disease. Cyclooxygenase-2 (COX-2) inhibition has emerged as a potential adjuvant to established treatment regimens for SCCHN with this concept in mind, as COX-2 is known to be overexpressed in SCCHN. Celecoxib is a COX-2 specific NSAID with known antineoplastic activity against many human tumors, and is currently being used in clinical trials as a chemopreventive agent and adjuvant to established chemotherapy and radiation therapy protocols. The underlying mechanism of celecoxib toxicity remains poorly understood. Previous work from our laboratory has demonstrated marked inhibition of cell cycle progression through the G1 phase and induction of apoptosis following treatment with celecoxib in SCCHN, leading to cell cycle phase-specific toxicity to S and G2 phase cells and induction of p21waf1/cip1 with downstream inhibition of nuclear E2F activity. We therefore hypothesize that decreasing the observed cell cycle inhibition induced by celecoxib treatment may augment celecoxib toxicity by inducing cells to progress through more highly toxic cell cycle phases during celecoxib exposure. Further understanding of the mechanism of G1 arrest following celecoxib administration is therefore needed to effectively exploit this potential toxicity. MicroRNAs have been implicated in the control of cell cycle transition in several recent publications in stem cells and human cancers, and we believe that they may be involved in the cell cycle arrest seen after celecoxib treatment in SCCHN. To evaluate this hypothesis, SCCHN cells were exposed to celecoxib for 24 hours and G1 arrest was confirmed via flow cytometry. RNA and protein were extracted from celecoxib-treated and control cells and microRNA expression profiles were established by quantitative real-time PCR microarray analysis. Alterations in cell cycle gene expression across the cell cycle were assessed by similar microarray, and targets were confirmed by further western blotting and PCR analysis. Correlations between microRNA alterations in celecoxib-treated cells and cell cycle control genes were observed and extrapolated. These data provide a microRNA and cell cycle gene expression signature for celecoxib treatment in SCCHN, and further illustrate the potential role that microRNA expression plays in control of G1 cell cycle checkpoint kinetics and subsequent celecoxib toxicity.

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 3009.
Abstract 3014: Robust microRNA expression profiling of specific cells in complex archival tissue stained by immunohistochemistry
Authors: Hlubek Falk , Schuster Claudia , Budczies Jan , Kirchner Thomas
Published: February 2011

Molecular diagnostics in modern pathology has been limited by the use of formalin-fixed, paraffin-embedded (FFPE) tissues in current routine diagnostic procedures due to modification and degradation of nucleic acids. In particular, molecular analysis of specific cell types potentially important for basic research and biomarker identification is largely prevented in highly complex, solid tissues routinely used in histopathology. Our objective was to establish a sensitive and robust procedure to quantify miRNA expression in specific cells from complex archival colorectal tumor tissue identified by immunohistochemistry. We combined classical immunohistochemistry and isolation of specific tissue cells by lasermicrodissection with the molecular analysis based on miRNA-preamplification and expression analysis by quantitative real-time PCR. Here we show reliable detection of miRNA expression profiles determined from limited amounts of colorectal cancer FFPE-tissues after routine staining procedure. The combination of routinely used FFPE-specimens stained by immunohistochemistry with the molecular analysis of lasermicrodissected complex tumor tissue resulted in robust miRNA expression patterns exclusively obtained from epithelial tumor cells. This approach allows for a detailed molecular analysis of cancer cells and distinct stromal cell types and their in-situ interaction in solid tumors. The methodology can therefore offer new perspectives for basic research and, by comprehensive use of present archival tissue collections linked to clinical databases, facilitate miRNA biomarker identification in defined tissue cells for future diagnostic and therapeutic strategies.

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 3014.
Abstract 3016: The value of microRNAs in the detection and staging of colorectal cancer
Authors: Ilyayev Nadia , Stojadinovic Alexander , Halle David , Roistacher Marina , Pavlov Vera , Mitrani-Rosenbaum Stella , Nissan Aviram
Published: February 2011

Introduction: MicroRNAs (miRNAs) are a large class of single strand RNA molecules consistent of 18-25 nucleotides, involved in post transcriptional gene silencing. miRNAs show tissue-specific expression and play an important role in carcinogenesis.

The aim of the present study was: A. to identify colorectal cancer (CRC)-specific miRNAs. B. To study the potential value of CRC-specific miRNAs in CRC detection and staging.

Methods: This study was approved by the Institutional Review Board (IRB). RNA was extracted from paired samples of normal and tumor tissue of CRC patients (n=10). miRNA expression was tested using a commercially available array. Differential expression between normal and tumor tissues was identified in 30 human miRNAs and in additional 16 miRNAs identified from database mining. Differential expression was validated by quantitative (real-time) PCR in a larger cohort of samples (n=20). RNA was extracted from sentinel lymph nodes (SLN) of CRC patients. A calibration plot was generated using a CRC cell line (HT29) mixed with peripheral blood lymphocytes (PBL) of healthy donors in a range of concentrations.

Results: After validation, only 8 miRNAs were found to be overexpressed in CRC.

The detection threshold for all 8 miRNAs measured by the calibration plot was 1:1000 (one HT-29 cell in 1000 PBMCs). The expression of the 8 miRNAs was then tested in SLNs (n=86). Fourteen lymph nodes (16.3%) were positive by standard pathological examination (H&E staining) and 25/86 (29.1%) were positive by miRNA expression analysis (p<0.01). miRNA expression was detected in all sera from CRC patients (n=3) and in none of the sera obtained from healthy individuals (n=5, p<0.0001).

Conclusions: A panel of 8 miRNAs was found to be overexpressed in CRC patients. Occult residual disease was indentified in lymph nodes and serum of CRC patients using this miRNA panel.

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 3016.
Abstract 3017: MicroRNAs as prognostic indicators in gastric cancer
Authors: Kushnir Michal , Hoshen Moshe B. , Purim Ofer , Meiri Eti , Cholakh Hila , Barabash Naama , Goren Yaron , Marshak Gideon , Kundel Yulia , Morgenstern Sara , Halpern Marisa , Niv Yaron , Chajut Ayelet , Brenner Baruch
Published: February 2011

Background: Our ability to determine the prognosis of an individual patient with gastric cancer and thereby to select patients for adjuvant therapy is still limited. Preliminary data suggest that microRNAs may have a prognostic role in this disease. We hence decided to compare the microRNA profiles in the primary tumor of patients with recurrent and non-recurrent gastric cancer and to evaluate the prognostic impact of these molecules.

Patients and Methods: Eligible patients, who underwent curative gastrectomies between 1995 and 2005 and did not receive any pre- or postoperative adjuvant therapy, were identified from the database of the participating institutions. Total RNA was extracted from tumor formalin-fixed paraffin embedded (FFPE) samples using proprietary protocols that preserve the small RNA fraction. Initial profiling using microRNA microarrays identified potential microRNA biomarkers that can be used to predict recurrence of gastric cancer after resection. The expression of differential microRNAs, was verified by qRT-PCR.

Results: Forty-five eligible patients had adequate tumor content and microRNA expression data and were included in the analysis. Of these, 14 (31%) experienced recurrence of disease within three years from surgery (“bad prognosis” group) and 31 (69%) did not (“good prognosis” group). Advanced pathological stage and extensive surgical procedure correlated with bad prognosis. Signal levels of three microRNAs were found to be significantly differentially expressed in tumors from patients with good prognosis vs. patients with bad prognosis. High expression of each of these microRNAs was associated with significantly poorer prognosis for both recurrence and survival. Proprietary microRNA qRT-PCR showed a high correlation to microarrays and similar separation into prognosis groups based on microRNA expression.

Conclusion: We identified a set of microRNAs whose high expression was predictive of tumor recurrence and poor prognosis in patients with gastric cancer. This finding provides a basis for a novel tool for risk stratification in this disease and allows further insight into its pathogenesis. Based on these results, a validation study, on a larger and independent cohort of patients, is planned.

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 3017.
Abstract 3025: Detection of prostatic fluid micro-RNA species associated with prostate cancer
Authors: Lewis Holly B. , Lance Raymond S. , Drake Richard R. , Kerscher Aurora E.
Published: February 2011

There is a need to develop more accurate and specific biomarkers that can be used in the early detection and determination of aggressiveness of prostate cancers. Collection of expressed prostatic secretions (EPS) in urine can be done following digital rectal exam (DRE) and gentle massage of each side of the prostate gland. This massage stimulates the release and movement of prostatic fluids and detached epithelial cells into the urethra. EPS is currently being used to develop genetic screens for expression of PCA3 non-coding RNA and TMPRSS2/Erg fusion transcripts, and thus could also provide a rich source of prostate-derived microRNAs (miRNAs). This collection is easily obtained, not disruptive to a standard urological exam and can be collected from men at all stages of prostate cancer, thus making EPS an ideal material to develop diagnostic screens for prostate cancer. Proteins and nucleic acids obtained from these fluids are characteristic of the prostate because the fluid is largely devoid of seminal vesicle-derived proteins or sperm. An additional prostatic fluid from cancer patients, termed direct EPS, has been collected via the penis following a vigorous pre-prostatectomy exam while the patient is under anesthesia. Our laboratory has begun to profile the expression of EPS-derived miRNAs to identify novel prostate cancer biomarkers. Large sample numbers of pre-DRE and post-DRE EPS urine specimens from prostate cancer patients and non-cancer controls have been collected to facilitate identification of cancer-specific as well as prostate-specific miRNA biomarkers. We have optimized procedures to isolate and profile the expression of EPS-derived miRNAs using stem-loop RT followed by TaqMan PCR analysis from 0.2-0.5 mls of sample. Several miRNAs linked to cancer progression (i.e., let-7, miR-16, miR-17, miR-21) have been successfully profiled and show differential expression patterns in post-DRE EPS urine samples obtained from prostate cancer patients compared to non-cancer controls. For comparison, we have also identified a subset of miRNA genes that are enriched in prostate cancer cell lines exhibiting invasive metastatic characteristics as well as miRNAs that are enriched in non-invasive prostate cell lines. We are testing if the expression of these miRNA subclasses correlates with the disease states of our patient EPS specimens, with the goal of developing miRNA signatures that could be used as new diagnostic and prognostic biomarkers for 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 3025.
Abstract 3027: Plasma microRNA as a potential marker for breast cancer detection
Authors: Ng Enders K.O. , Leung Candy , Au Stephanie , Chan Aaron , Wong Chris , Ma Edmond , Pang Roberta , Chua Daniel , Chu KM , Law WL , Poon Ronnie TP , Kwong Ava
Published: February 2011

Background: Recently, the emergence of small non-protein-coding RNAs, so-called microRNAs (miRNAs), playing important roles in oncogenesis has opened new opportunities for early cancer diagnosis. We have previously shown that microRNAs (miRNAs) in plasma are promising biomarkers for colorectal cancer detection. Here, we investigated whether plasma miRNAs could discriminate patients with and without breast cancer (BC).

Methods: Using TaqMan-based low density miRNA array, miRNAs were profiled from cancerous and adjacent non-cancerous breast tissues, corresponding plasma of 5 BC patients, along with plasma from 5 healthy controls. Marker selection and validation were performed by real-time quantitative RT-PCR on a small set of plasma. Independent set of plasma from 80 BC patients, 20 gastric cancer, 20 lung cancer, 20 colorectal cancer, 20 hepatocellular carcinoma and 50 healthy controls were further validated.

Results: Of the panel of 377 miRNAs analyzed, 8 miRNAs (miR-16, miR-21, miR-27a, miR-141, miR-191, miR-200c, miR-210, miR-451) were up-regulated both in plasma and tissue samples of 5 BC patients. All 8 putative miRNA markers were validated on the plasma of 15 BC patients and 15 healthy controls. Only three miRNAs were significantly elevated in this cohort of BC patients (p<0.0005). The plasma levels of the three markers in those 15 BC patients were significantly reduced after surgery (p<0.05). Further validation with an independent set of plasma samples (n=210) indicated that two markers differentiate BC from normal subjects, colorectal cancer, gastric cancer, hepatocellular carcinoma and lung cancer. These two markers yielded a combined receiver operating characteristic curve area of 88.5%, the sensitivity was 92% and the specificity was 72% in discriminating BC from control subjects.

Conclusions: Plasma miRNAs significantly elevated in BC patients are identified. This can be a novel noninvasive molecular marker for BC screening.

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 3027.
Abstract 3031: Screening for plasma micro RNAs as biomarkers for breast cancer detection
Authors: Marian Catalin , Bebu Ionut , Kella Danesh , Zhou Xin , Islam Md , Ressom Habtom , Shields Peter , Zheng Yun-Ling
Published: February 2011

Introduction: MicroRNAs (miRNAs) are small, non-coding RNA molecules that play critical roles in a variety of physiological and pathological processes, including carcinogenesis of breast cancer. The research regarding miRNA discovery and characterization is emerging and there is consensus in the literature that miRNAs are excellent candidate biomarkers for human cancer. Cancer specific miRNAs have been detected in tumors as well as in serum, plasma, and other body fluids. However, no extensive profiling of circulating miRNAs in breast cancer patients has been done to date, although several studies investigated a limited number of miRNA panels.

Methods: We investigated the miRNA profile in plasma samples collected before surgery from 10 pathologically confirmed breast cancer patients, one month after surgery from 2 breast cancer patients, and from 10 healthy women controls that were frequency matched to cases on age and race. RNA was extracted from plasma using the mirVana Paris RNA isolation kit (Ambion) and miRNA profiling was done using the TaqMan® Human MicroRNA Array Set v.2.0 (Applied Biosystems), enabling the detection of 667 human mature miRNAs. Plasma miRNA levels were calculated using the well established comparative Ct method (ΔΔCt) relative to the selected controls, and both Wilcoxon rank-sum tests and t tests were used to compare plasma miRNA levels between control subjects and cancer patients. Correlation between miRNAs was assessed separately in cancer and control subjects.

Results: Ten miRNAs (1.5% of total) were detected in plasma of all subjects and 12 miRNAs (1.8% of total) were detected in at least 80% of cases and controls, respectively. Of these, 17 miRNAs presented a significant differential expression in plasma between cases and controls (p<0.05). Only one of these (has-miR-191) was previously described as being differentially expressed in breast cancer tissue compared to normal breast tissue. Based on their ability to discriminate between cancer and controls, their fold change, p values, correlation coefficients and post-surgical levels, we selected 6 candidate miRNAs that will be validated in a larger sample set of 50 cases and 50 controls. The p values of these six candidates ranged between <0.0001 and 0.005 and the fold change between cases and controls ranged from 33.85 to 111.80.

Conclusion: We identified several circulating miRNAs that are differentially expressed between breast cancer patients and normal controls, and therefore could serve as biomarkers for breast cancer detection if confirmed by future studies.

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 3031.
Abstract 3033: Profiling of global changes in microRNA levels during adipocyte differentiation by real-time PCR
Authors: Sebastian Thomas , Dhundia Elica , Yerramilli Subrahmanyam , Lader Eric
Published: February 2011

We have used a novel real time PCR approach to profile global changes in microRNA expression during adipocytic differentiation of 3T3-L1 fibroblast cells. This profiling strategy makes use of a novel two stage screen resulting in a substantial increase in throughput and decrease in reagent use. In the first step, cDNA is prepared from individual samples of undifferentiated fibroblast, and cells at early and late stages of Adipocyte differentiation. A pooled cDNA sample prepared by pooling equimolar amounts of individual cDNAs was used in an initial profiling experiment to identify microRNAs that are expressed at detectable levels in the experimental system. Profiling was performed in triplicate with real time PCR assays for 591 mouse miRNAs (miRBase v12.0). In the second stage of screening, individual cDNA samples were each analyzed in triplicate using the assays for miRNAs that showed expression in the pooled cDNA sample. Expression data was normalized against a panel of small non-coding RNAs and was expressed as x-fold changes relative to expresion in undifferentiated 3T3L1 cells. Our results indicate that 192 (∼32%) miRNAs described in miRBase v12.0 were detectable in resting and differentiated 3T3-L1 cells, and of these, 38 (or 20%) showed differential regulation during adipogenesis (x-fold change>3.0). When compared to data from previous array-based profiling studies, our screen identified additional differentially regulated miRNAs. In addition to profiling changes in mature miRNA levels, miRNA precursor stem loop transcripts were also quantified to gain insight into post transcriptional regulation (if any) of miRNA biogenesis during adipocyte differentiation. The high specificity and sensitivity of the miScript RT-PCR system, together with a novel sample-pooling strategy makes this system a method of choice for miRNA expression profiling. Pooled primary profiling can substantially reduce the total number of PCR reactions which need to be performed and increases assay throughput.

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 3033.
Abstract 3039: microRNA profiling of gastric cancer patients from formalin-fixed paraffin-embedded (FFPE) samples
Authors: Shimada Yutaka , Okumura Tomoyuki , Osawa Soshi , Sekine Shin-ichi , Moriyama Makoto , Sawada Shigeaki , Nagata Takuya , Takahashi Hiroyuki , Fukuoka Junya , Takano Yasuo , Fukumoto Manabu , Tsukada Kazuhiro
Published: February 2011

InIntroduction: microRNA (miRNA) are small non-coding RNAs which target specific mRNA. Recent progression of RNA extraction technology from formalin-fixed paraffin-embedded (FFPE) tissues enables us to access miRNA profiling using world wide stored specimens. Here, we present miRNA profiling of gastric cancer patients from FFPE specimens.

Method: First, we examined the level of cross-linking caused by formalin and degradation of mRNA and miRNA from day to year levels. Then, we established the criteria of evaluable RNA from FFPE specimens. The miRNA profiling was obtained from 47 gastric cancer patients who received operations from 1997 to 2007 using 3D-Gene human miRNA chip (Toray) which contains 885 miRNA species.

Results: Out of 47 paired samples, 37 pairs (78.8%) were determined evaluable with our criteria. As results, we showed that 30 miRNAs were significantly down-regulated and that 15 miRNAs were up-regulated in gastric cancer in comparison to normal gastric tissue. Of these, 18 miRNAs, including mir-21 and mir-34a, were identified as a prognostic factor of gastric cancer patients. Furthermore, we found 25 miRNAs have association with the recurrence of adjuvant oral Tegaful treatment and 5 miRNAs have association with lymph node metastasis.

Conclusion: We have discovered miRNAs that are associated with prognosis, chemosensitivity and lymph node metastasis in gastric cancer patients. miRNA profiling using FFPE specimens is a useful and promising method of evaluation of world wide stored specimens that accompany may valuable clinical data.

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 3039.
Abstract 3040: Differential microRNA expression between normal edometrium, endometrioid adenocarcinoma, papillary serous adenocarcinoma, and carcinosarcoma
Authors: Devor Eric J. , Goodheart Michael , DeGeest Koenraad , Bender David , Buekers Thomas , Leslie Kimberly K.
Published: February 2011

This study compares quantitative microRNA expression profiles of normal endometrial tissue and the three primary endometrial tumor types; endometrioid adenocarcinoma, papillary serous adenocarcinoma, and carcinosarcoma, representing a spectrum of tissue involvement, aggressiveness, and prognosis/ recurrence risk.

Methods: Total RNA was prepared directly from primary tissues and microRNA expression was profiled in two ways. Expression levels of 667 human microRNAs were assessed by qPCR using TaqMan microRNA Arrays (Applied Biosystems). In addition, a size-selected small RNA cloning strategy developed in our lab was employed to search for additional microRNAs via deep sequencing on the Illumina/Solexa platform.

Results: A number of recent surveys have implicated a small set of candidate microRNAs in endometrial cancers. Among theses are miR-103, miR-107, miR-141, miR-210, as well as the miR-200 family, particularly miR-200c. Our results confirm these previous studies as well as implicating several additional microRNAs.

Conclusions: Quantitative microRNA expression profiling of endometrial cancers demonstrates differential expression that segregates with tumor type and clinical prognosis. In addition, a number of specific microRNAs appear to be consistently either over- or under-expressed in these cancers compared to normal endometrium. It is these microRNAs, such as miR-210 and miR-200c, that comprise a set of markers for quantitative validation and target ascertainment with the potential to provide important correlates to cancer type and prognosis/ recurrence risk.

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 3040.
Abstract 3041: Differential expression of microRNAs between sporadic and hereditary nonmedullary thyroid cancer
Authors: Xiong Yin , Kebebew Electron
Published: February 2011

Background: MicroRNAs (miRNAs) are small noncoding RNAs, which regulate gene expression and play a significant role in many biological processes, including tumorigenesis. Approximately 5% of all non-medullary thyroid cancers are hereditary and may have more aggressive tumor biology than sporadic cases. The susceptibility gene for hereditary non-medullary thyroid cancer has not been identified. We hypothesized that miRNA expression profile between sporadic and hereditary non-medullary thyroid cancer are different and could help identify candidate suspceptibility genes.

Methods: Twenty nine tumor tissue samples were analyzed using miRNA microarrays for 1190 miRNAs with genotyping for common somatic mutations (BRAF V600E, NRAS, KRAS, RET/PTC1, RET/PTC3) present in non-medullary thyroid cancer. The samples were matched for age, gender and stage of cancer. Differences in miRNA expression profile were validated by quantitative RT-PCR and correlated with clinicopathologic data.

Results: We found 5 miRNAs significantly differentially expressed between sporadic and hereditary non-medullary thyroid cancer cases; two miRNAs were upregulated and three miRNAs were downregulated in hereditary non-medullary thyroid cancer. Two miRNAs were validated by quantitative RT-PCR; miR-20a (p=0.025) and miR-886-3p (p=0.028) were upregulated in hereditary non-medullary thyroid cancer samples. Higher expression level of miR-886-3p was significantly associated with higher TNM stage (p<0.05). There was no significant difference in somatic mutation rates between sporadic and hereditary cases (9 BRAF V600E, 1 RET/PTC1), and there was no significant difference in miR-20a and miR-886-3p expression by mutation status.

Conclusions: To our knowledge, this is the first study to show different expression profile of miRNAs between sporadic and hereditary cancer using non-medullary thyroid cancer as a model. These differences in miRNAs may have an important role in tumor cell biology and clinical manifestation of non-medullary thyroid cancer in sporadic versus hereditary cases.

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 3041.
Abstract 3044: Enzymatic bioluminescence assay for microRNA quantification
Authors: Sun Ye , Golovlev Val
Published: February 2011

A quantitative bioluminescence assay (qBLA) for rapid and sensitive microRNA expression analysis was developed. The qBLA detects the release of inorganic pyrophosphate (PPi) when target microRNA hybridizes with its complementary oligonucleotide template and is extended by DNA polymerization reaction. The luminescence detection of PPi is performed by converting PPi to ATP by ATP-sulfurylase that provides energy for luciferase to oxidize luciferin and produce light. This approach is faster, simpler, and less expensive than microarrays and real-time PCR for microRNA detection. It requires less reagents and can be performed at a constant temperature without thermal cycling. The experimental results indicate a broad dynamic range for microRNA quantification. Strategies for isothermal amplification of microRNA targets with different oligonucleotide template designs were studied, which illustrate the improved sensitivity and specificity. In this report, the real-time and end-point microRNA detection by qBLA is presented and discussed.

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 3044.
Abstract 3045: Identification of specific microRNA signatures in VHL-functional and VHL-silenced renal cell carcinoma cell lines
Authors: Kurisetty Vittal , JeBailey Lellean , Luongo Tony , Merchan Jaime R.
Published: February 2011

Objective& Background: Renal cell carcinoma is the most common renal malignancy in adults. Up to 70% of clear cell renal cancers have VHL silencing either because of gene mutation or methylation. MicroRNAs are a class of small non-coding RNAs that control gene expression by targeting mRNAs and trigger either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases including cancer. This study set out to investigate the influences of VHL status and the ensuing differences in miRNA profiles and biological/phenotypic effects in renal carcinoma cell lines. Methods: Normal cell line, HREC, and renal cancer cell lines that are VHL-ve cell lines, A498, A704, 786.O, and 786.O. Vec, VHL+ve cell line,786.O.VHL, VHL-WT cell line, ACHN and Caki.1 and VHL-CH3 cell line, KV6 were used for the VHL-specific genome wide miRNA profiling. miRNAs were extracted, in triplicate, from the above cell lines at 48 hrs time point by using miRVana miRNA isolation kit (Ambion). The extracted miRNAs were subjected to analysis by MEGAPlex RT primers and MEGAPlex Pre-Amp primers, followed by loading them to TaqMan Human miRNA microarrays v2.0 (Human Array A/B (Applied Biosystems). Resulting miRNAs were analysed further by using the RealTimeStatMiner v3.0 software (Integromics) followed by GeneGO pathways analysis. Results: We show that approximately 577 miRNAs were altered in human kidney cancer. 270 miRNAs and 180 miRNAs were upregulated and down-regulated in cancer cell lines when compared against normal HREC cell line, respectively. Upon analysis of differentially expressed miRNAs in RCC cell lines based on VHL status, we found that 7 miRNAs were differentially upregulated in VHL wild type RCC cells. In VHL mutant cell lines, 5 miRNAs and 6 miRNAs were differentially up and down regulated, respectively, while in RCC cell lines with wild type, but silenced (methylated) VHL, 7 and 10 miRNAs were differentially up and down regulated, respectively. Target prediction and microRNA enrichment analysis suggested that the differentially expressed miRNAs are involved in regulation of HIF, angiogenesis, invasion & metastasis, extracellular matrix degradation, EMT phenomenon, metabolism and apoptosis pathways. Conclusion: This MicroRNome analysis reveals a specific spectrum of hypoxia, angiogenesis, tumor metabolism, metastasis, cell cycle and apoptotic specific miRNAs. In vitro experiments to characterize the biological significance differential miRNA expression profiles on different steps of tumorigenesis and tumor progression are underway.

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 3045.
Abstract 305: Frequent PIK3R1 somatic mutations promote oncogenic signaling
Authors: Jaiswal Bijay S. , Janakiraman Vasantharajan , Chaudhuri Subhra , Stern Howard M. , Kljavin Noelyn M. , Wang Weiru , Kan Zhengyan , Bowman Krista , Wu Jiansheng , Kaminker Joshua , Yue Peng , de Sauvage Frederic J. , Backer Jonathan , Seshagiri Somasekar
Published: February 2011

We performed a systematic analysis of human fresh frozen tumors for somatic mutations in the RAS-effector pathways and have identified frequent mutations (>8%) in PIK3R1 (p85α) regulatory subunit of PI3K (p110) enzyme complex. Mutations in other members of the PI3K regulatory subunits, along with mutations in additional components of the pathway were also identified in this study. An integrated analysis of somatic mutations and genomic copy number variations identified by comparative genome hybridization revealed distinct mutation patterns across tumor types. In order to understand the functional consequence of p85α mutations, we generated mutant p85α constructs and tested them for activity in biochemical and cellular assays. In this studies, we found that all the mutants that contain an intact p110-binding domain were able to interact with p110α, p110β and p110δ. Also, all these mutants stabilized expression of p110 class IA members when tested in pan-p85 null fibroblasts, indicating that the p110-stabilizing activity of p85 mutants was still intact. However, in un-stimulated cells, some of the p85α mutants were not as effective as wild-type p85α in inhibiting the activity of p110. Furthermore, these p85α somatic mutants activated AKT signaling leading to cell survival. Additionally, cells expressing the p85α mutants when transplanted in vivo promote a leukemia-like disease leading to decreased overall survival of the mice compared to wild-type p85α. These data demonstrate an alternate mechanism for activation of class IA PI3Ks in tumors through somatic mutations in p85α that abrogates its p110 regulatory activity while maintaining its p110-stabilizing activity. The frequent occurrence of p85α mutations and its ability to promote survival signaling suggest that tumors with such mutations are likely candidates for inhibitors that target the components of the PI3K pathway.

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 305.
Abstract 3054: MicroRNA expression profile in cutaneous melanoma: miR-17-92 cluster is a progression and prognostic marker
Authors: Kim Donghwa , Zhao Jianjun , Kong William , Smalley Keiran S. , Messina Jane L. , Weber Jeffrey S. , Cheng Jin Q.
Published: February 2011

MicroRNAs (miRNA) represent a novel class of RNAs that play important roles in cell survival, proliferation, differentiation and angiogenesis through negative regulation of protein-coding genes. Deregulation of miRNAs has been detected in various types of human malignancies. Some miRNAs are implicated in diagnosis, prognosis, treatment response and pathogenesis. However, aberrant miRNA expression and its pathologic significance in human melanoma have not been well documented. Here, we report on the miRNA expression profile of human cutaneous melanoma as well as melanoma cell lines. Of 650 miRNAs examined, at least 28 miRNAs were significantly deregulated in melanoma compared to normal skin and primary melanocytes, with the most common aberrantly expressed miRNAs being miR-17-92 cluster, miR-21, miR-146a/b. miR-148a/b, miR-126, miR-606, and miR-655. Notably, miRNA expression signatures could stratify patients into 2 groups (e.g., good and poor prognosis). Further analysis revealed that elevated levels of the miR17-92 cluster are associated with tumor progression and shorter overall survival. Our results suggest that miRNAs may be a useful new biomarker for and be a therapeutic target in cutaneous melanoma.

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 3054.
Abstract 3055: Wnt inhibitory factor 1 inhibits cervical cancer cell growth in vitro
Authors: Ramachandran Ilangovan , Ramalingam Satish , Natarajan Gopalan , Mills Wilbur K. , Anant Shrikant , Reis Antonio M.C. , Queimado Lurdes
Published: February 2011

Background and Purpose: Cervical cancer is one of the most common malignancies and major causes of death in women worldwide. Aberrant activation of Wnt/β-catenin pathway is associated with various cancers including cervical cancer. Mutations within intracellular components of the Wnt pathway are rare in cervical cancer. We hypothesize that alterations in secreted Wnt antagonists might play a major role in Wnt activation. Here we characterize the status of the Wnt inhibitory factor 1 (WIF1) in cervical cancer cell lines and determined the in vitro effects of WIF1 on cervical cancer cells. Methods: Three cervical cancer cell lines (HeLa, C33A and CC1) were treated with the demethylating agent 5-aza-2′-deoxycytidine (DAC) for 4 days, total RNA was isolated and real-time RT-PCR was performed to determine WIF1 mRNA expression. HeLa cells were transfected simultaneously with either pCI blast (empty vector) or pCI blast-WIF1 using LipoD293 transfection reagent and assessed for cell proliferation and apoptosis. Cell proliferation was determined at different time intervals (24, 48, 72 and 96 h) by hexosaminidase assay. Cell cycle analysis was performed after 72 h by flow cytometry using FACSCalibur analyzer. Caspase 3/7 activity was measured using the Apo-one Homogeneous Caspase-3/7 Assay kit. Results: We demonstrated that WIF1 is down-regulated in cervical cancer cells. Treatment with DAC caused a significant increase in WIF1 mRNA expression in all the cell lines tested compared with vehicle treatment. Furthermore, transfection with a plasmid expressing WIF1 significantly inhibited cervical cancer cell proliferation at all time points studied. To probe the possible mechanisms involved in WIF1 cell growth inhibition, we performed cell cycle analysis and caspase 3/7 assay. Interestingly, cell cycle analysis demonstrated that transfection with WIF1 significantly increased the G2-M cell population and the number of apoptotic cells. In addition, WIF1 significantly induced caspase 3/7 activity suggesting that WIF1 induced apoptosis is mediated through caspase 3/7 activation. Conclusions: Our data suggests that WIF1 is silenced in cervical cancer cells and WIF1 restoration inhibits cervical cancer cell growth by inducing G2-M arrest and apoptosis. This study emphasizes the importance of WIF1 as a potential therapeutic strategy in the treatment of cervical 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 3055.
Abstract 3058: ADAMTS9 inhibits angiogenesis and induces apoptosis in nasopharyngeal carcinoma
Authors: Lung Hong Lok , Lo Paulisally Hau Yi , Cheung Arthur Kwok Leung , Apte Suneel S. , Lung Maria Li
Published: February 2011

Background and aims: A disintegrin-like and metalloprotease with thrombospondin type 1 motif 9 (ADAMTS9), a gene mapping to 3p14.2, has been shown to be silenced by promoter hypermethylation in nasopharyngeal carcinoma (NPC). The ADAMTS family consists of 19 secretion protein members consisting of a disintegrin domain, metalloproteinase domain, and different numbers of thrombospondin type 1 motifs. ADAMTS9 encodes a member of a large family of 19 metalloproteases involved in maturation of precursor proteins, extracellular matrix remodeling, cell migration, and inhibition of angiogenesis. Although the related matrix metalloproteases and ADAM proteases have been clearly implicated in tumor progression and angiogenesis, the role of ADAMTS proteases in cancer is less clearly defined. In the present study, we investigated the in vivo and in vitro functional roles of ADAMTS9 in angiogenesis of NPC.

Methods: To examine the antigiogenic activity of ADAMTS9 in NPC, the in vivo gel plug and in vitro tube formation assays were performed using a tetracycline-regulated expression vector in the present study.

Results: Here we show that when incubated with conditional media from the ADAMTS9 stable transfectant, the human umbilical vein endothelial cells (HUVEC) showed a dramatic decrease of tube formation ability. When the gel plugs used for the in vivo angiogenesis in the above section were stained with H&E, we observed that the numbers of apoptotic cells in the ADAMTS9 stable transfectant were dramatically increased. The numbers of apoptotic cells were also increased when growing in the absence of serum. Conclusions: Taken together, these studies indicate that ADAMTS9 is an important candidate tumor suppressor gene in NPC, which might mediate its tumor suppressive effects through the anti-angiogenic and pro-apoptotic mechanisms.

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 3058.
Abstract 3061: Tumor suppressive and angiogenic role of THSD1 in esophageal squamous cell carcinoma and nasopharyngeal carcinoma
Authors: Ko Josephine Mun Yee , Cheung Arthur KL , Lung Maria Li
Published: February 2011

Background and aims: Loss of chromosome 13q regions is a frequent event in both esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). By microcell-mediated chromosome transfer, chromosome 13 was demonstrated to play a significant tumor suppressive role in both ESCC and NPC in our laboratory. THSD1 is a novel candidate tumor suppressor gene (TSG) previously identified by microarray differential gene expression profiling. It encodes a transmembrane molecule containing a thrombospondin type 1 repeat (TSR), which may be involved in cell adhesion and angiogenesis. Proteins possessing the TSR, such as thrombospondin-1 (Tsp1), ADAM metallopeptidase with thrombospondin type 1 motif, 1 (ADAMTS1) and ADAMTS8, regulate angiogenesis. We previously showed that the mechanisms for THSD1 silencing in ESCC involved loss of heterozygosity and promoter hypermethylation. Our preliminary data indicated transfection of wild type THSD1 into an ESCC cell line, SLMT-1, resulted in significant reduction of colony-forming ability, hence providing functional evidence for its growth suppressive activity. The current study aims to examine if THSD1 plays a significant tumor suppressive role in the nude mouse tumorigenicity assay and to elucidate the mechanism(s) involved in tumor suppression. Methods: Transfection by lipofection of both variants of THSD1 into the ESCC SLMT-1 and NPC HONE1-2 cell lines was used to study the effect of THSD1 in tumor suppression and anti-angiogenesis. Results: Quantitative RT-PCR detected down-regulation of THSD1 expression in 36.4% (16/44) primary ESCC and 54.5% (12/22) NPC tissues. Nude mouse tumorigenicity assays showed tumor suppression in THSD1 over-expressing clones in both ESCC and NPC. Flow cytometry analysis showed over-expression of THSD1 resulted in endoreplication. Tube formation assay with HUVEC is underway to examine the angiogenic role of THSD1. Conclusions: THSD1 is a promising candidate TSG in both ESCC and NPC.

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 3061.