bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2040: Identification of microRNAs associated with tamoxifen resistance in breast cancer
Authors: Lau Lai-yee , Chan Kelvin Yuen-kwong , Khoo Ui-Soon
Published: February 2011

Tamoxifen is the most commonly used endocrine therapy for estrogen receptor-positive breast cancer patients. As an estrogen antagonist, tamoxifen competes for binding with estrogen receptor, thereby inhibiting estrogen-dependent gene transcription and tumor growth. However, some breast tumors become estrogen-independent and ultimately develop resistance to the treatment. The mechanisms underlying this transition in the control of cell proliferation remain unclear. Recent work has shown that aberrant expression of microRNAs has been frequently detected in breast cancer and some of which are associated with breast cancer metastasis, poor prognosis, suggesting an important role of microRNAs in breast cancer development and progression. Certain microRNAs negatively regulate AIB1/ACTR, ERα, HER2 and HER3 and deregulation of these genes are well documented to modulate the resistance to tamoxifen in breast cancer. Therefore, we hypothesized that deregulated microRNAs might confer the aberrant expression of their target genes and the development of tamoxifen resistance in breast cancer.

To identify microRNAs that might confer resistance to tamoxifen in breast cancer, we examined expression profiles of more than 600 microRNAs in a pair of tamoxifen-sensitive ZR75 and -resistant AK47 breast cancer cell lines using TaqMan Low Density Array (Applied Biosystems). Sixty-five microRNAs were identified substantially downmodulated in the tamoxifen-resistant cell line while 44 microRNAs were found to be upregulated. Consistent with others’ previous findings, our profiling results indicated that miR-221 and miR-222 were overexpressed in the tamoxifen-resistant breast cancer cells. Of the differentially expressed microRNAs, miR-449a and miR-449b were significantly downregulated in the resistant AK47 cells. miR-449a/b expression was also examined in 60 frozen breast tumors by TaqMan Individual MicroRNA Assay (Applied Biosystems). The expression of miR-449a/b was inversely correlated with tumor grade (miR-449a: OR=0.11, 95%CI=0.03-0.38, p<0.001; miR-449b: OR=0.23, 95%CI=0.07-0.70, p=0.008) and strongly associated with estrogen receptor status of the tumor (miR-449a: OR=5.57, 95%CI=1.67-18.55, p=0.003; miR-449b: OR=5.0, 95%CI=1.51-16.56, p=0.006). Our results suggest that miR-449a/b may influence tamoxifen sensitivity in breast cancer cells. Further functional studies are being performed to investigate the regulatory roles of the microRNAs in the development of tamoxifen resistance in breast 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 2040.
Abstract 2041: PDGF inducible microRNAs regulate gene expression in cancer cells
Authors: Shao Minghai , Rossi Simona , Chelladurai Bhadrani , Ntukogu Obiageli , Ivan Mircea , Calin George , Matei Daniela
Published: February 2011

Background: Platelet derived growth factor (PDGF)-regulated signaling plays a critical role in oncogenesis. By binding to the PDGF receptors, the Akt, Src and MAPK pathways are activated to regulate genes expression and cellular physiological processes. There are subtle functional differences between the effects of specific PDGF ligands (e.g. AA, BB), which are incompletely understood. MicroRNA(miR)s are single-stranded, 21-23 nucleotide regulatory RNAs that repress target gene expression either by inhibiting protein translation or by promoting target mRNA degradation. The hypothesis tested in this study was that PDGFs regulate genes expression by modulating microRNAs level in cancer cells.

Methods: MicroRNA microarrays were performed to identify miR alterations after treatment of U118 glioblastoma cells with PDGF-AA and BB. Several time points were tested: 2, 6, 12, and 24 hour. Significance Analysis of Microarrays (SAM) was performed to compare expression level between different time points. Q-PCR was conducted to confirm the microarrays data. Pre-miR probe/miRCury LNA probe were transfected into cancer cells to over-express/knock down selected miRs. Western blots were used to detect miRs’ target proteins.

Results: Candidate PDGF-inducible/repressible miRs were identified based on significant change in expression level after AA/BB treatment (p-value <0.01). Expression levels of 7 miRs were altered by PDGF AA and expression levels of 14 miRs were altered after PDGF BB treatment. By Q-PCR we showed that let 7d was repressed by PDGF-AA, but not by PDGF-BB in cancer cells (U118, C272 and U87) and in fibroblasts (WI38). Cyclin D1 is a known target of let7d. By overexpression and knock-down strategies we show that PDGF-AA induced cyclin D1 expression is mediated via repression of let7d. Q-PCR confirmed that miR-146b and miR-106b are inducible by PDGF-BB, but not by PDGF-AA in cancer cells (U118, C272 and U87) and in fibroblasts (WI38). MiR-146b induction by PDGF-BB is modulated via MAPK-dependent induction of Fos. A Fos binding element was identified in the promoter region of miR146b and Fos binding to this region in the presence of PDGF-BB was confirmed by chromatin immunoprecipitation. Several targets of miR146b are altered by PDGF in cancer cells.

Conclusions: Our data demonstrate for the first time that PDGF signaling regulates expression of several critical oncogenic miRs which in turn alter target gene expression and function.

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 2041.
Abstract 2043: Inhibition of cervical carcinoma cells proliferation by miR-34c
Authors: Lopez Jesus A. , Salas Luis M. Alvarez
Published: February 2011

MicroRNAs (miRNAs) play central roles in controlling cell proliferation, differentiation, and apoptosis. Aberrant miRNA expression is recognized as an important molecular mechanism for human carcinogenesis. Infection with high-risk human papillomaviruses (HPVs) is causally associated with the onset of cervical dysplasia including cancer. Although high-risk HPV infection leads to hyperproliferation and immortalization, it is insufficient to produce a fully transformed phenotype. High-risk HPV E6 proteins (i.e. HPV-16 and 18) inactivate and destroy P53 protein leading to a p53-null phenotype and the subsequent repression of the p53 effector network including pro-apoptotic proteins and cell cycle inhibitors. Microarray analyses in normal and tumor cervical cells have linked only a few miRNAs to cervical cancer. However, no miRNAs have been established in the malignant process that may follow a high-risk HPV infection. Here, we analyze the function of miR-34c (a p53 effector) in HPV-positive cells. Steady-state miR-34c levels are very low in both immortal and tumor HPV-positive cells. Over-expression of miR-34c in cervical carcinoma cells caused inhibition of cell proliferation and anchorage independent growth resulting in G2/M arrest and significant apoptosis. Interestingly, no effect was noticed in HPV-negative tumor cells and HPV-immortalized keratinocytes suggesting a differential regulatory pathway of mi-34c in HPV-positive tumor cells. Analyses of miR-34c potential targets showed no significant effect on BCL-2 levels although it produced MAP-2 down-regulation. These results suggest that activation of miR-34c in the absence of functional p53 may play an important role in proliferation and apoptosis of HPV-positive cervical tumor cells that could be used as therapeutic or diagnostic/prognostic tool for cervical cancer therapy.

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 2043.
Abstract 2046: The hepatic miR-200/Zeb module is perturbed in the male Fischer rat following short term treatment with a carcinogenic dose of Phenobarbital
Authors: Koufaris Costas , Wright Jayne , Currie Richard A. , Gooderham Nigel J.
Published: February 2011

Few studies have so far examined the role of microRNAs (miRNAs) in directing the adaptive or adverse responses of the liver following chemical carcinogen exposure. Nevertheless, significant evidence supports the importance of miRNAs in normal liver functioning and their dysregulation in diseased states.

In this study we chose to look at the effect of Phenobarbital, a prototype rodent non-genotoxic hepatocarcinogen, on liver miRNAs of the male Fischer rat. The animals were exposed to either a carcinogenic (1000 ppm) or two non-carcinogenic (50 and 500 ppm) dietary doses of Phenobarbital for up to 90 days. Microarrays and quantitative Real-time PCR were then used to profile liver miRNA expression.

Unsupervised hierarchical clustering separated the samples according to dose after 14 days, that is the carcinogenic dose of Phenobarbital resulted in a distinct miRNA profile. A number of miRNAs showed statistically significant changes in expression uniquely to the carcinogenic dose at 14 days. Prominent was miR-200b, which was specifically upregulated by the carcinogenic dose of Phenobarbital atfter 14 days, but not before then. Real-time PCR confirmed this upregulation of miR-200b and that it was maintained after 90 days exposure to Phenobarbital. Mir-200b is a member of the miR-200 family of miRNAs, which form part of a negative feedback loop with two protein targets: Zeb1 and Zeb2. Western blots confirmed that both proteins are downregulated specifically by the carcinogenic dose of Phenobarbital.

This study demonstrates that carcinogenic doses of Phenobarbital cause distinct effects on miRNA expression compared to non-carcinogenic doses, specifically an alteration in the miR-200/Zeb module was observed. Importantly, others have reported perturbation of this module in the liver of rats following carcinogenic treatments with acetoaminofluorine and choline-deficient L-amino Acid Defined diet. Collectively these data suggest that the miR-200/Zeb module could be an important element in the response of the liver to chemical carcinogen exposure.

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 2046.
Abstract 2048: microRNA regulation of tissue factor expression in breast cancer cells
Authors: Zhang Xiao-Xi , Yu Haijun , Lou Jessica R. , Zheng Jie , Popescu Narcis-Ioan , Lupu Florea , Ding Wei-Qun
Published: February 2011

Tissue factor is recognized to be an important regulator of tumor angiogenesis and metastasis, independent of its participation in the blood coagulation pathway. The tissue factor gene is highly expressed only in breast carcinoma cells having high invasive potential, but the mechanisms regulating tissue factor gene expression in breast cancer cells remain unclear. This study examined the hypothesis that microRNA regulation of tissue factor expression is responsible for over-expression of the tissue factor gene in invasive breast cancer cells. Tissue factor was highly expressed in the highly invasive MDA-MB-231 line but was barely detectable in the less invasive MCF-7 line, as assayed by RT-PCR and western blot. A reporter gene assay showed that the tissue factor promoter can be activated in extracts prepared from MCF-7 cells. Forced expression of tissue factor cDNA was achieved in MCF-7 cells, implying that that tissue factor expression is regulated by the 3′-UTR of its transcript. Bioinformatics analysis suggested that microRNA species such as miR-19 (miR-19a and 19b) and miR-106b interact with the 3’-UTR of the tissue factor transcript and may regulate tissue factor protein translation and/or mRNA degradation. microRNA array confirmed that miR-19b is highly expressed in MCF-7 cells, as compared to MDA-MB-231 cells. A reporter gene assay using a TF-3′-UTR-luciferase construct indicated that the tissue factor 3′-UTR negatively regulates tissue factor expression, an effect that was completely reversed by deletion of the miR-19 binding site in the reporter construct. Application of an antisense microRNA for miR-19 enhanced tissue factor expression in MCF-7 cells. This study demonstrates that tissue factor expression in breast cancer cells is regulated by miR-19. The expression pattern of miR-19 in breast cancer tissues and its role in mediating angiogenesis and metastasis merit future investigation.

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 2048.
Abstract 2058: Translational regulation of SPARC by the microRNAs mir29a, b, and c
Authors: Trieu Vuong , Hwang Larn , Lin Xiaoqin , Tamir Rami , Motamed Kouros , Desai Neil
Published: February 2011

Background: SPARC (Secreted Protein Acidic and Rich in Cysteine) is overexpressed in most cancers, including breast, prostate, lung, brain, head-and-neck, and kidney. SPARC overexpression enhances tumor progression, angiogenesis, and metastasis and is generally associated with poor prognosis. In a previous study, SPARC mRNA was detected among the majority of both normal and cancer tissues using RT-PCR, in contrast to immunohistochemistry (IHC) that showed SPARC protein only in tissue remodeling and in cancers. These results suggested a post-transcriptional regulation of SPARC mRNA. We report here microRNA (miRNA) mediated regulation of SPARC mRNA.

Methods: A chimera expression vector with CMV driven Luciferase ORF upstream of SPARC's 3′UTR was constructed and used in an assay to detect miRNA mediated suppression of luciferase translation. The construct was co-transfected into a permissive cell line- HEK293 (embryonic kidney cells) and individual miRNA mimics or inhibitors obtained from Dharmacon (Lafayette, Co, USA). Inhibitors of luciferase ORF were detected by a suppression of the luciferase signal. Deletion analysis on SPARC's 3′UTR was also performed to localize sequence required for translational suppression. Findings using CHO and 293 cells were confirmed using other cell lines including ES-2, SKOV-3, CAOV-3, PA-1, U87-MG, and OVCAR-3.

Results: It was noted during forced expression of SPARC that CHO cells did not support SPARC overexpression, whereas HEK293 could. Screening using the luciferase ORF linked to SPARC's 3′UTR revealed that mir29 a, b and c were able to cause translational inhibition of SPARC mRNA. Deletion mutants of 3′UTR from SPARC restored the luciferase activity, whereas inhibition was retained when region 1-236 nt of SPARC's 3′UTR was maintained suggesting that the mir29 binding sequence is located within this region. The finding was not specific to CHO, and mir29 inhibition of SPARC expression was confirmed among all other cell lines tested (ES-2, SKOV-3, CAOV-3, PA-1, U87-MG and OVCAR-3).

Conclusions: Human mir29a, b and c down-regulated the SPARC translation across a number of cell lines tested. These data suggest that that miRNAs mir29 a, b and c could play an important role in tumor metastasis and progression by control of SPARC expression.

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 2058.
Abstract 2066: Role of miR-182/miR-96 cluster in prostatic zinc homeostasis
Authors: Khramtsova Ekaterina A. , Vaishnav Avani , Arva Nicoleta , Antonio Angeline , Kajdacsy-Balla Andre , Nonn Larisa
Published: February 2011

Zinc has been shown to have a defensive action against the development of prostate cancer (PCa). Zinc is an essential element that is required for the activity of more than 300 enzymes, structure of proteins, and control of genetic expression. It plays an important role in cellular processes such as cell division, growth, differentiation, development, aging, and synthesis and repair of DNA, RNA, and protein. Prostatic secretary epithelial cells accumulate large amounts of zinc and have a high expression of human Zinc transporter 1 (hZIP1), the major zinc transporter in the prostate. PCa tissue exhibits lower levels of zinc and hZIP1 compared with surrounding normal-appearing areas. Furthermore, compared to Caucasian men, the prostates of African American men, a population with a disproportionately high risk of developing PCa, have decreased zinc and hZIP1. Previously, we examined several miRNAs in formalin-fixed paraffin-embedded prostate tissue from African American and Caucasian patients, of which miR-182/96 levels were higher in PCa samples and had inverse correlation with hZIP1 mRNA in Caucasian patients (Spearman rho = −0.77, p=0.009). No correlation between hZIP1 and miR-182 was found in the African-American specimens, suggesting a different mechanism for hZIP1 regulation in African-Americans. Based on previous studies, we hypothesize that miR-182/96 cluster is responsible for regulation of hZIP1 levels in PCa and prostate tissue. In this study, we examined the relationship between the levels of miR-182/96 and hZIP1 in more detail using cell culture models. We observed an inverse correlation between hZIP1 and miR-182 levels in several prostate cell cultures; primary normal epithelial cells, normal stromal cells, and prostate cancer cells (PC3 and LNCaP). MiR-182 is transcribed in a cluster with miR-96 and the miRNAs have an overlapping mRNA target sequence. As expected, miR-96 expression was identical to that of miR-182, thus also inversely correlated with hZIP1 in prostate cell cultures. Overexpression of miR-182 in both normal and LNCaP cells decreased hZIP1 mRNA levels, further implicating miR-182 as a regulator of zinc transport. Ongoing experiments focus on validating the two putative miR-182/96 binding sites in the 3′UTR of hZIP1 and the effect of miR-182 and miR-96 overexpression on zinc transport. Our results may establish a role for miR-182 in prostatic zinc homeostasis. Because zinc levels are linked to PCa risk, these miRNAs may be useful as a prognostic or diagnostic marker in prostate biopsies to identify patients with high risk of PCa recurrence and for identifying individuals for zinc replacement.

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 2066.
Abstract 2067: Effects of microRNA-34a on brain tumor cells and glioblastoma stem cells
Authors: Guessous Fadila , Zhang Ying , Catania Alessia , Li Yunqing , Kofman Alexander , Kefas Benjamin , Schiff David , Purow Benjamin , Abounader Roger
Published: February 2011

Glioblastoma and medulloblastoma are the most common malignant brain tumors in adults and children, respectively. microRNAs are deregulated in different types of cancer including brain tumors. We previously found that microRNA-34a (miR-34a) inhibits brain tumor growth by targeting multiple oncogenes. We showed that miR-34a is downregulated in glioblastoma tumors. miR-34a down-regulated c-Met in human glioblastoma and medulloblastoma cells and Notch-1, Notch-2, and CDK6 protein expressions in glioblastoma cells. miR-34a expression inhibited c-Met reporter activities in glioblastoma and medulloblastoma cells and Notch-1 and Notch-2 3′-UTR reporter activities in glioblastoma cells and stem cells. miR-34a strongly inhibited cell proliferation, cell cycle progression, cell survival, and cell invasion in glioblastoma cells, but not in astrocytes. Forced expression of c-Met or Notch-1/Notch-2 transcripts lacking the 3′-UTR sequences partially reversed the effects of miR-34a on cell cycle arrest and cell death in glioblastoma cells and stem cells, respectively. Also, in vivo transient expression of miR-34a in glioblastoma cells strongly inhibited intracranial xenograft growth. We therefore showed that miR-34a suppresses brain tumor growth by targeting c-Met and Notch. We subsequently investigated the effect of miR-34a on glioblastoma stem cell malignancy and differentiation. We transfected 0308 and 1228 glioblastoma stem cells with 30 nM pre-miR-34a or control miR for 72 hours and assessed the cells for cell cycle status, cell proliferation, cell migration and apoptosis by propidium iodide flow cytometry, cell counting, scratch migration assay and annexin V flow cytometry, respectively. We also assessed the effect of miR-34a on stem cell differentiation into neurons, astrocytes or oligodendrocytes, by immunohistochemistry staining for CD133, nestin and differentiation markers including Claudin, GFAP, and Tuj-1. miR-34a slightly inhibited cell proliferation in both stem cell lines. miR-34a also induced G1/S cell cycle arrest. Moreover, miR-34a restoration led to a slight increase in stem cell death in both stem cell lines and also strongly inhibited migration in 1228 stem cells. Importantly, miR-34a treatment of stem cells reduced CD133 staining and increased differentiation marker staining. These data suggest a role for miR-34a in the regulation for glioblastoma stem cell malignancy and differentiation. Supported by a University of Virginia Cancer Center Pilot Project Grant.

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 2067.
Abstract 2072: Systematic functional analysis of microRNAs by transfection of 1129 miRNAs into prostate cancer cells
Authors: Ostling Paivi , Leivonen Suvi-Katri , Aakula Anna , Kohonen Pekka , Makela Rami , Perala Merja , Kallioniemi Olli
Published: February 2011

The molecular mechanisms behind prostate cancer development, progression and therapy failure are incompletely understood, but the androgen receptor (AR) is almost invariably involved. Altered microRNA (miRNA) expression profiles have recently been demonstrated in a large number of cancer types, including prostate cancer. These short RNA molecules of ∼21 bases regulate mRNA stability and translation by base-pairing mainly to the 3’-untranslated region (3′UTR) of their targets.

In this study, we analyzed the functional impact of miRNA transfections on prostate cancer cell growth, survival, and expression of the AR protein. Seven prostate cell lines (LNCaP, LAPC-4, MDA-PCa-2b, 22Rv1, CWR-1R, RWPE-1, and EP156T) were screened with 1129 miRNA mimics (Ambion, Dharmacon). Cell viability was measured with CellTiter-Glo® (CTG, Promega) whereas AR, cell proliferation (Ki67) and apoptosis (cPARP) were detected with specific antibodies using protein lysate microarrays as described in our recent publication on estrogen receptor targeting miRNAs (Leivonen et al., Oncogene 2009). Seventy-one of the miRNAs had anti-proliferative or pro-apoptotic functions when transfected to the prostate cancer cell lines. In addition, 17 of these miRNAs down-regulated the AR protein. Western blots were used to verify knockdown, and the direct functional role of the miRNAs was clarified by 3′UTR assays and by analyses of down-stream consequences of the knockdowns.

In summary, we have systematically defined those miRNAs that are critical for prostate cancer growth, survival and the expression of the androgen receptor. This integrated, multi-parametric profiling provides new evidence of the role of miRNAs in prostate cancer as well as novel clues for therapeutic targeting by manipulating key pathways and cell survival mechanisms by miRNA transfections.

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 2072.
Abstract 2077: MicroRNA signature of hyperplastic enlarged lobular units (HELU) from patients with high risk of breast cancer
Authors: Nilsson Sofie , Landberg Göran
Published: February 2011

In order to detect early pathological changes in breast tissue, which could reveal the underlying mechanism to cancer progression, we investigated the microRNA (miRNA) expression profile in hyperplastic enlarged lobular units (HELUs). HELUs arise from normal terminal duct lobular units (TDLUs) and are potentially the earliest histologically identifiable alterations in the development of breast cancer.

TDLUs and HELUs, from formalin-fixed paraffin embedded tissue from patients with high risk of developing breast cancer, were laser capture microdissected and the RNA was extracted. Expression profiles were obtained using a qRT-PCR-based array analyzing 667 human miRNAs.

In the first set of miRNA array analyses of paired samples from four specimens with early HELUs, we obtained a list of 16 downregulated and 10 upregulated miRNAs with a significant change (p<0.05) in expression. Among the most pronounced altered miRNAs were miR-136, miR-301b, miR-302b and miR-576-5p.

To validate these results, more paired samples of fully developed HELUs will be added to the study and candidate miRNAs will be further validated using qPCR and in situ hybridization of formalin fixed specimens using a selection material of 100 patients with high risk of breast cancer. These ongoing miRNA analyses will potentially reveal additional miRNAs that are linked to the development of TDLUs towards HELUs as well as substantiating the frequency of the various alterations observed.

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 2077.
Abstract 2084: Tumor suppressor microRNA miR-34 inhibits human pancreatic cancer stem cells
Authors: Tang Wenhua , Ji Qing , Hao Xinbao , Li Ling , Jeffrey DeSano , Fearon Eric R. , Lawrence Theodore S. , Xu Liang
Published: February 2011

MicroRNAs (miRNAs) have been implicated in cancer initiation and progression via their ability to affect expression of genes and proteins that regulate cell proliferation and/or cell death. Transcription of the three miRNA miR-34 family members was recently found to be directly regulated by p53. We show that miR-34 directly regulates Notch and Bcl-2, suggesting the possibility of a role for miR-34 in the maintenance and survival of cancer stem cells.

We examined the roles of miR-34 in p53-mutant human pancreatic cancer cell lines and the potential link to cancer stem cells. Restoration of miR-34 expression in the cancer cells by miR-34 mimics or lentiviral miR-34 downregulated Bcl-2 and Notch1/2. miR-34 restoration significantly inhibited clonogenic cell growth and invasion, induced apoptosis and G1 and G2/M arrest in cell cycle, and sensitized the cancer cells to chemotherapy and radiation. We identified that CD44+/CD133+ cancer cells are enriched with tumorsphere-forming and tumor-initiating cells or cancer stem/progenitor cells with high levels of Notch/Bcl-2 and loss of miR-34. More significantly, miR-34 restoration led to an 87% reduction of the tumor-initiating cell population, accompanied by significant inhibition of tumorsphere growth in vitro and tumor-initiation in vivo. Our results demonstrate that miR-34 may restore, at least in part, the tumor suppressing function of the p53 in p53-deficient human pancreatic cancer. Our data support that miR-34 may be involved in cancer stem cell self-renewal, potentially via the direct modulation of downstream targets Bcl-2 and Notch, implying that miR-34 may play an important role in cancer stem cell self-renewal and/or cell fate determination. Currently, we are exploring the nanoparticle-targeted delivery of miR-34 to cancer stem cells as a novel molecular therapy for human pancreatic cancer, based on our patented nanobiotechnology which has been approved by FDA for clinical trials. Taken together, our results demonstrate that restoration of miR-34 may hold significant promise as a novel molecular therapy for human pancreatic/gastric cancer with loss of p53-miR34, potentially via inhibiting cancer stem cells. The project is funded by NIH grants CA121830, CA128220 and CA134655 (to L. Xu.).

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 2084.
Abstract 209: Gamma linolenic acid sensitizes pancreatic cancer cells to chemotherapy
Authors: Espinoza Ingrid C. , Busby Robert , Lupu Ruth
Published: February 2011

In pancreatic cancer both chemotherapy and radiotherapy have shown deficient results in treating the disease, indicating a fundamental need for more efficient therapies. Most pancreatic cancer patients present with an advanced form of the disease that is incurable by surgery, and will receive palliative treatment in the form of surgery, chemotherapy, radiotherapy and symptom control. There are few standard chemotherapeutics for the treatment of pancreatic cancer, although Gemcitabine and 5-Fluorouracil (5-FU) are the most widely used. In search for specific nutritional, and/or pharmacological agents to target pancreatic cancer cells/tissues we identified a series of nutritional supplements known as polyunsaturated fatty acids (PUFAs) that have an inhibitory effect on cancer cells. However, Gamma-Linolenic-Acid (GLA), a member of the ω-6 family of PUFAs exerts selective cytotoxic effects on pancreatic cancer cells without affecting normal epithelial cells. The goal of our study was to determine whether GLA could sensitize the targeted pancreatic cancer cells to chemotherapeutic agents (Gemcitabine or 5-FU). Therefore, pancreatic cancer cells were treated initially with GLA followed by each of the chemotherapeutic agents separately: Our results demonstrated that: 1) GLA induced growth inhibition and apoptosis of pancreatic cancer cells (Panc-1 and BxPC3); 2) the combination of GLA and Gemcitabine or GLA and 5-FU: a) promote a synergistic inhibitory effect on the growth of pancreatic cancer cells; b) inhibition of anchorage-independent growth; and 4) inhibition of morphogenesis in 3-D matrigel culture. These results show that GLA increases the sensitivity of pancreatic cancer cells to chemotherapy through a synergistic effect with Gemcitabine or 5-FU, implying that GLA could be used as a supportive drug for the pancreatic cancer patients receiving the current chemotherapeutic regimens.

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 209.
Abstract 2099: Induction of tumor suppressor genes IL24 and IL32 by microRNA-205 in prostate cancer
Authors: Majid Shahana , Dar Altaf A. , Saini Sharanjot , Chen Yi , Shahryari Varahram , Liu Jan , Zaman Saif , Hirata Hiroshi , Yamamura Soichiro , Ueno Koji , Tanaka Yuichiro , Khatri Gaurav , Carroll Peter R. , Dahiya Rajvir
Published: February 2011

Introduction and Objective: MicroRNAs are small noncoding RNAs that regulate the expression of approximately 30% of all human genes, either inhibiting target mRNA translation or inducing its degradation. These genes are abnormally expressed in human malignancies, making their biological importance increasingly apparent. Given the functional complexity of miRNA mediated gene regulation, it is unlikely that miRNAs are limited to gene silencing. The main objective of this study was to investigate the involvement of miRNA-205 in prostate carcinogenesis and whether miR-205 functions as a tumor suppressor by transcriptionally up-regulating tumor suppressor genes IL24 and IL32.

Methods: The methods employed in this study include quantitative-real time PCR; western blot; in-situ hybridization; fluorescence-activated cell sorting assays for cell cycle distribution and apoptosis; assays for cell viabiltity, clonability, migration and invasiveness of prostate cancer cells. Luciferase reporter and mutational assays; nuclear run-on, in-vitro transcription and chromatin immunoprecipitation assays were also performed.

Results: Our results indicated that miR-205 expression levels are either silenced or significantly lower in prostate cancer cell lines and prostate tumors when compared to non-malignant prostate cell lines or benign prostate hyperplasia tissues respectively. By scanning gene promoters for sequences complementary to miR-205, we identified putative miR-205 target sites in the promoters of IL24 and IL32 tumor suppressor genes. Over-expression of miR-205 in prostate cancer cells (PC-3) induced the expression of both genes by targeting specific sites in their promoters. Transfection of miR-205 impaired cancer cell growth, clonability, migration, invasiveness and induced apoptosis and cell cycle arrest by interfering with the expression of genes involved in these pathways. MicroRNA-205 induced in-vitro transcription and caused enrichment of histone markers indicative of transcriptionally active promoters in IL24 and IL32 genes. Luciferase reporter and mutational assays indicated that IL24 and IL32 are the direct targets of miR-205.

Conclusion: For the first time we have shown that microRNA-205 specifically activates tumor suppressor genes IL24 and IL32 by targeting complimentary binding sites in their promoters, corroborating a new function for miRNAs in regulating gene transcription. Such a function of microRNAs may contribute to the novel therapeutic approach in the treatment 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 2099.
Abstract 2102: Exogenous microRNA-101 suppresses cell proliferation by downregulation of cyclooxygenase-2 expression in prostate cancer cell line
Authors: Hao Yubin , Pang Xiaowu , Zhao Yuan , Califano Joseph A. , Gu Xinbin
Published: February 2011

MicroRNAs, ∼22 nt non-coding small RNAs, have been newly focused as a potential cancer diagnostic biomarker and therapy target, because aberrant expressions of miRNAs have been often found to involve in tumorigenesis, cancer development and progression. For instance, microRNA-101 (miR-101) is significantly down-regulated in several types of cancer. In this study, we investigated the function of miR-101 on regulation of gene expression in cancer cells. A stable miR-101 over-expressed prostate cancer cell line (BPH1CAFTDmiR101) was established by using lentivirus-delivery system. Over-expressed miR-101 level in the stable transfected cell can be detected after the 5th or 100th cell culture passages and approximately 10-fold higher than non-transfected BPH1CAFTD cells. We found that over-expressed miR-101 suppressed cyclooxygenase-2 (COX-2) protein expression in BPH1CAFTDmiR101 cells. The miR-101 targeted gene was determined by using luciferase reporter gene, which carry 3’-UTR (NM_000963 3’-UTR 1735-1741) of COX-2 mRNA. The results indicated that miR-101 directly targets COX-2 gene and down-regulates COX-2 protein expression by binding to 3’-UTR of COX-2 mRNA. Interestedly, the reduced COX-2 protein expression in BPH1CAFTDmiR101can be restored by transient transfection of COX-2 3′UTR fragment. Compared with BPH1CAFTD cells, BPH1CAFTDmiR101 proliferation and colony formation were significantly inhibited, and the expressions of a series of cell proliferation-related protein were significantly changed, such as the levels of EGFR, PCNA, and Bcl-2 were decreased and the level of p53 was increased. In addition, MMP-15 protein level, a metastasis related protein, was also noticeably reduced. Taking together, our finings demonstrate that miR-101 inhibits prostate cancer cell proliferation by directly targeting COX-2 to down-regulating its protein level. The miR-101 may function as tumor suppressor and a potential target for cancer diagnosis and therapy.

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 2102.
Abstract 2103: miR-140 and miR-192 are potential tumor suppressors through inhibiting cellular proliferation and restoring cell cycle control in breast cancer cell lines
Authors: Song Bo , Karaayvaz Mihriban , Ju Jingfang
Published: February 2011

Abstract

MicroRNAs (miRNAs) are a class of small non-coding RNAs in regulating expressions of multiple mRNA targets mostly by binding at the 3’-UTRs to inhibit mRNA translation or facilitate mRNA cleavage. MiRNAs are involved in the control of many fundamental cellular and physiological processes and mounting evidence indicates that miRNAs play important roles in human cancers acting either as oncogenes or tumor suppressor genes through their global impact on multiple genes and pathways. Previous studies from our group have discovered two important miRNAs, miR-140 and miR-192, in suppression of cellular proliferation and triggering cell cycle arrest in both colon cancer and osteosarcoma cell lines. In this study, we further elucidate the molecular and cellular impact of these two miRNAs in breast cancer. miR-140 or miR-192 were over-expressed by transient transfection in a panel of breast cancer cell lines such as MCF-7 and MDA-MB-231. The proliferation of MCF-7 cells transfected with miR-140 or miR-192 was dramatically decreased at 100 nM while the control miRNA has no effect. Inhibition of proliferation by miR-140 or miR-192 was also observed in MDA-MB-231 cells but less potent than MCF-7 cells at the equal concentration. MCF-7 cells transfected with miR-140 or miR-192 were arrested at G2/M phase of the cell cycle. The cell cycle check point control genes p53 and p21 were induced by over-expressing miR-140 or miR-192. The endogenous miR-140 or miR-192 expression was induced in MCF-7 cells treated with methotraxate (MTX) accompanied with increased expression of p53. Blocking endogenous miR-140 or miR-192 by locked nucleic acid (LNA) modified anti-miRNA increased cellular proliferation rate in MCF-7 cells. In conclusion, miR-140 and miR-192 may have therapeutic potential in breast cancer.

Keywords: microRNA, breast cancer, miR-140, miR-192

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 2103.
Abstract 2111: Genomic rearrangements in BRCA1 and BRCA2 loci in Chilean breast cancer families
Authors: Carvallo Pilar , Sánchez Alejandro , Faúndez Paola , Camus Mauricio , Alvarez Manuel
Published: February 2011

Mutations in BRCA1 and BRCA2 are responsible of about 20% of hereditary breast cancer in Chilean families. Studies in breast cancer families from other populations have identified the amplification and/or deletion of one or more exons in these genes, as the cause of the disease. In the present study we determined the presence of these types of alterations in BRCA1 and BRCA2, in 76 Chilean families, non carriers of BRCA1/BRCA2 mutations, by using MLPA analysis (Multiple Ligation-dependent Probe Amplification). We identified 2 different alterations in the BRCA1 gene: in one family 1.6 times amplification of exon 10 was found, and amplification of exons 3, 5 and 6 was detected in 3 families, with ratios 1.5; 5.5 and 4.5. These alterations, confirmed in a second MLPA analysis, have not been previously described in other populations. To validate the MLPA results and to characterize these genomics alterations we performed qPCR, long range PCR and sequencing. Amplification of exon 10 was confirmed by qPCR, but sequencing was not possible due, in part, to the presence of several repeats in the adjacent introns. In the two unrelated patients showing amplification ratios of 5.5 and 4.5 for exons 3, 5 and 6, qPCR results were concordant with amplification of only exon 5. Long range PCR and sequencing of these gene fragments revealed the presence of exon 5 duplicated and contiguous to exon 6. The third unrelated patient showing amplification of exons 3, 5 and 6 with a ratio of 1.4, did not show differences with control DNA in qPCR analysis. This study reveals the necessity to confirm MLPA results with qPCR and/or sequencing of the involved gene fragments to validate exonic amplifications. Financed by Fondecyt 1080595.

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 2111.
Abstract 212: Targeting p73 as a strategy for enhancing anticancer efficiency of chemotherapeutic drugs to triple-negative human breast cancers
Authors: Yu Weiping , Tiwary Richa , Kline Kimberly , Sanders Bob
Published: February 2011

Successful treatment of p53-mutant, triple-negative breast cancer (TNBC) remains a daunting challenge. Chemotherapeutic drugs such as doxorubicin (DOX) and cisplatin (CDDP) remain the first line treatments for TNBC; however, chemotherapy eventually fails due to acquired drug resistance and toxicity. Therefore, new treatments for overcoming drug resistance and toxicity in TNBC are badly needed. Here we report on the reconstitution of the p53 tumor suppressor pathway in a p53-independent manner via p53 homolog p73 as an exciting possibility for improving cancer therapy in p53 mutant breast cancers. α-TEA, a small bio-active lipid analog of vitamin E, exhibits potent anticancer actions in human breast and murine mammary cancer both in vitro and in vivo. Treatments with combinations of α-TEA plus DOX or CDDP enhances in vitro apoptotic killing of two p53-mutant TNBC cells; namely, MDA-MB-231 and BT-20 cells. Sub-apoptotic doses of α-TEA plus non-apoptotic doses of DOX or CDDP significantly enhanced caspase-8 and 9 mediated apoptosis in both cell lines. The combination treatments enhanced p73 protein expression, enhanced levels of death receptors-5 (DR5), CD95/APO-1 (Fas), pro-apoptotic Bax and Noxa proteins, and reduced pro-survival Bcl-2 protein levels, all of which are p53 regulated genes. The combination treatments induced enhanced levels of phospho-c-Ab1 and JNK, upstream mediators for p73. Furthermore, combination treatments activated Yes-associated protein (Yap), a transcriptional coactivator which has been demonstrated to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Taken together, our data suggest that combination of α-TEA with clinically relevant chemotherapeutic drugs (DOX or CDDP) enhances apoptosis of MDA-MB-231 and BT-20 p53-mutant TNBC cells via p73, and that c-Abl/JNK and Yap may contribute to the enhanced anti-cancer efficacy of the combination treatments. These data provide the rationale for conducting pre-clinic TNBC xenograft studies testing the safety and efficacy of this new therapeutic approach. Supported by DOD concept grant W81XWH-09-1-0564 (Yu).

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 212.
Abstract 2124: The analysis of EGFR genetic heterogeneity on mixed adenocarcinoma of the lung
Authors: Tomonaga Nanae , Nakatomi Katsumi , Nakamura Yoichi Nakamura , Ogawara Daiki , Senju Hiroaki , Ikeda Takaya , Yamaguchi Hiroyuki , Doi Seiji , Iida Tetsuya , Hayashi Tomayoshi , Nagayasu Takeshi , Tsukamoto Kazuhiro , Kohno Shigeru
Published: February 2011

Background: The epidermal growth factor receptor thyrosine kinase inhibitors (EGFR-TKIs) are effective for non-small cell lung cancer (NSCLC) patients harboring sensitive EGFR mutations. However, every patient eventually relapse even if the treatment of EGFR-TKIs was effective remarkably. It have been reported a secondary T790M mutation, MET amplification and some mechanisms attribute to the drug resistance. Intratumor EGFR genetic heterogeneity has also been considered as a potential cause of the acquired resistance. We previously reported a patient with mixed adenocarcinoma of the lung that had different EGFR mutations in papillary, acinar and BAC subtypes. In this study, we analyzed EGFR mutations in each histological subtypes of mixed adenocarcinoma after surgery and pre EGFR-TKIs treatment.

Material and Methods: We recruited twelve mixed adenocarcinoma patients who were treated surgical therapy. Tumor cells in different histological subtypes were microdissected from paraffin-embedded surgical specimens with AS LMD system. Genomic DNA was extracted from each sampled area. EGFR mutations, limited to exon19 deletions and L858R mutations, were examined using the simple polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) methods.

Results: Acinar, BAC and papillary sanmples were microdissected from six, ten and eleven tumor tissues. Exon19 deletions and L858R mutation were detected in five and one tissues. From acinar, BAC and papillary samples, one exon19 deletion, two exon19 deletions and three exon19 delitions and one L858R were detected. Out of the six mixed adenocarcinoma, five tissues consisted both mutated and non-mutated cells. Conclusions: Our results indicates that 41% of lung adenocarcinoma with mixed subtypes have intratoumor genetic heterogeneity. EGFR mutations were detected most frequently in papillary samples. There may be some relations between histological subtype and EGFR mutation.

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 2124.