bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1116: Neurotensin high affinity receptor 1 is a functional biomarker for lung and breast cancer progression
Authors: Doan Van Kien , Zaïd Sadi Menad Ahmed , Younes Mohamad , Dupouy Sandra , Mourra Najat , De Wever Olivier , Alifano Marco , Gompel Anne , Forgez Patricia
Published: June 2012

Neurotensin (NTS) is a 13 amino acid peptide, exerting hormonal and neurocrine regulation within the gastrointestinal tract to globally facilitate nutriment digestion. NTS action is mainly mediated by the high affinity G protein coupled neurotensin receptor, NTSR1. Abnormal expression of NTSR1 occurs during the early stages of cell transformation in relation with the deregulation of the Wnt/beta catenin pathway. Consequently, the oncogenic effects previously described for NTS in different types of cancer cells could presumably contribute to tumor progression. In order to establish this concept, we observed that NTS and NTSR1 were concomitantly expressed in human breast ductal carcinomas and lung adenocarcinomas. NTSR1 was found to be an independent prognostic marker in lung and breast clinical studies. Cellular models were developed in which NTS or NTSR1 was either abolished by sh-RNA or over-expressed. In experimental tumors using both models, active NTS autocrine or paracrine regulation caused the enhancement of tumor size and metastasis emergence when compared to those having poor expression of NTS or NTSR1. In vitro studies, confirmed that NTS enhancement of cellular growth, adherence, motility and invasion occurred through the activation of PKC, ERK 1/2 pathways, and by interfering with the network of epidermal growth factor tyrosine receptors, as EGFR, HER2, and HER3 expression and activation were increased. The use of NTSR1 specific antagonist and neutralizing NTS antibody confirmed the contribution of NTS in cellular oncogenic effect implicated in each step of cancer progression. Additionally, the activation of metalloprotease induced by NTS contributed to the release to EGF-like ligand. Our results suggest that the activation of the NTSR1 pathway causes an acceleration of the oncogenesis process within preexisting tumors by the up regulation of the three majors epidermal growth factor tyrosine receptors and the formation of autocrine stimulation of the HERs system.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1116. doi:10.1158/1538-7445.AM2011-1116
Abstract 1123: Transforming growth factor α signaling in pancreatic acinar-to-ductal metaplasia
Authors: Liou Geou-Yarh , Storz Peter
Published: June 2012

Pancreatic cancer is the most lethal type of cancer due to its high proliferation, metastasis and resistance to chemotherapy. In pancreatic cancer, pancreatic ductal adenocacinoma (PDAC) is the most common form. Current opinion is that PDAC originates from acinar or centroacinar cells though a process called “acinar-to-ductal metaplasia” (ADM). During ADM, acinar cells undergo a transformation to ductal PanIN (pancreatic intraepithelial neoplasia) cells, which is believed as the first step in PDAC formation. Transforming growth factor α (TGFα) is produced by pancreatic cancer cells and promotes tumor growth. In an ex vivo explant culture system, TGFα transforms murine pancreatic acinar cells into ductal cells. Here we describe a cell signaling pathway that TGFα utilizes to induce acinar-to-ductal transformation. We show that K-ras is downstream of TGFα and is sufficient to drive ADM. We further show the mechanisms that K-ras uses to induce cell structural rearrangement of acinar cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1123. doi:10.1158/1538-7445.AM2011-1123
Abstract 1131: Differential induction of c-fms promoters by TGFβ in cervical cancer and macrophage cell lines
Authors: Kirma Nameer B. , Ganapathy Manonmani , Krishnegowda Naveen K. , De La Garza Elizabeth M. , Tekmal Rajeshwar R.
Published: June 2012

Cervical cancer is the second most common gynecological malignancy in the world. An expected 12,200 total new diagnosed cases were estimated by the American Cancer Society for 2010 (Cancer Facts and Figures, 2010). Human papillomavirus infection, a major cause for cervical cancer, results in a persistent and pathological inflammatory response, contributing to establishment of disease and malignant progression. Our previous studies have shown that inflammatory factors, like Cox2, tumor associated macrophages, and cytokines such as macrophage colony-stimulating factor (CSF-1) and transforming growth factor beta (TGFβ) are elevated in cervical carcinomas. The receptors for CSF-1 (encoded by the c-fms oncogene) and TGFβ were also elevated in cervical carcinomas compared to normal cervical tissue. Both CSF-1 and c-fms have been implicated in aggressive gynecological malignancies, including cervical cancer. CSF-1/c-fms signaling is involved in proliferation, differentiation and recruitment of macrophages as well as cancer cell growth. We have previously demonstrated that TGFβ induced the expression of c-fms mRNA in cervical cancer cell lines and macrophage cell line THP-1. Using flow cytometry in this study, we have confirmed induction of c-fms protein at the cell surface due to TGFβ treatment, suggesting that TGFβ may enhance CSF-1 signaling by inducing cell surface expression of its receptor. Our goal was to examine the mechanisms underlying the regulation of c-fms in macrophages, relevant leukocytes associated with inflammation and pro-malignant growth within the tumor microenvironment. Two promoters for the c-fms gene exist, promoter one driving expression in trophoblasts (trophoblast specific promoter) and promoter two driving expression in macrophages (macrophage specific promoter). Consistent with our previous findings, TGFβ induced c-fms expression via MAPK p38 mainly at the trophoblast specific promoter in cervical cancer cells, as shown by the use of the p38 inhibitor SB203580. Our data also suggest that Casein kinase 1 mediates c-fms expression via this promoter in cervical cancer cells. In the macrophage cell line THP-1, p38 mediated the expression of c-fms via both promoters, as demonstrated by using exon specific RT-PCR primer sets. The c-fms mRNA induced at the macrophage specific promoter is the major c-fms transcript in THP-1 cells and is likely to mainly contribute to c-fms expression in these cells. Understanding the regulation of c-fms in macrophages and cervical cancer cells may pave the way to targeting c-fms expression to interrupt aberrant CSF-1 signaling in cervical cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1131. doi:10.1158/1538-7445.AM2011-1131
Abstract 1133: LRIG2 promotes PDGF induced experimental glioma
Authors: Holmlund Camilla , Karlsson Terese , Borota Olivera Casar , Miller Jamie , Baofeng Wang , Zahed Farrah , Sweeney Colleen , Henriksson Roger , Hedman Håkan
Published: June 2012

Leucine-rich and immunoglobulin-like domains protein 2 (LRIG2) is a transmembrane protein, whose expression is associated with poor survival of oligodendroglioma patients. This is in contrast with findings for the homologous protein LRIG1, a negative regulator of growth factor signaling, whose expression is associated with good survival in several different malignancies. Here, we address the role of LRIG2 in an experimental glioma model and its possible regulation of growth factor receptors belonging to the epidermal growth factor receptor (EGFR) and platelet-derived growth factor (PDGF) receptor (PDGFR) families. Gliomas were induced in Ntv-a mice by intracranial injection of PDGFB-expressing RCAS viruses. All injected Lrig2-wild-type mice developed oligodendroglioma-like brain tumors of grade II/III (82%) or glioblastoma-like tumors of grade IV (18%), whereas Lrig2-deficient mice only developed grade II/III tumors (77%) or no detectable tumors (23%). A role for Lrig2 in PDGFR signaling was shown in mouse embryonic fibroblast (MEF) cell lines. MEF cells from Lrig2-deficient mice were less sensitive to PDGFB-induced cell proliferation than MEF cells from wild-type mice. In transfected HEK-293T cells, LRIG2 and LRIG3, in contrast to LRIG1, did not negatively regulate the protein levels of EGFR, ErbB2, PDGFRα, or PDGFRβ. Intriguingly, LRIG2 and LRIG3 instead up-regulated the protein levels of PDGFRα. In summary, we show that LRIG2 promoted the genesis and/or progression of PDGF-induced oligodendroglioma and positively regulated PDGFRα protein levels. This further supports the notion that LRIG proteins play important roles in cancer biology and suggests that LRIG1 and LRIG2/LRIG3 may have opposing functions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1133. doi:10.1158/1538-7445.AM2011-1133
Abstract 1134: ADAM17 promotes glioma cell progression through EGFR-PI3K-AKT activation
Authors: Zheng Xuguang , Jiang Feng , Katakowski Mark , Lu Yong , Chopp Michael
Published: June 2012

Background & Objective: A disintegrin and metalloproteinase-17 (ADAM17) is involved in proteolytic ectodomain shedding of several membrane-bound growth factors and cytokines. The expression and activity of ADAM17 increase under some pathological conditions such as stroke and cancer. ADAM17 promotes neural progenitor cell migration and contributes to stroke-induced neurogenesis after stroke and breast cancer growth and invasion. In the present study, we sought to elucidate whether ADAM17 contributes to glioma progression and its mechanisms.

Experimental Approaches: We examined the role of ADAM17 in the proliferation, invasion, and tube formation of human glioma cells (U87) in vitro and tumor growth in vivo. Stable transfection of the U87 cell line with either a plasmid for over-expression of human ADAM17, or a siRNA to ADAM17 was employed in this study to establish high or low ADAM17 expression in glioma cells, respectively. For study of mechanism, the ADAM17 inhibitor TAPI-2 and the PI3K-AKT inhibitor LY294002 were used to counteract high ADAM17 expression or the activated PI3K-AKT pathway. Proliferation of glioma cells were tested by MTT Cell Proliferation Assay, Bromodeoxyuridine incorporation assay, growth curve, and sulforhodamine B assay. Matrigel invasion assays were used to assess the ability of U87 cells to penetrate the Extra Cellular Matrix. A Matrigel tube formation assay was performed to test capillary tube formation ability. EGFR-PI3K-Akt pathway activation in U87 cells under different ADAM17 expression levels were tested by Western blot

Results: Our data show that ADAM17 promotes the U87 malignant phenotype by increased proliferation, invasion, angiogenesis and in vivo tumor growth. TGF-α, VEGF secretion and VEGF expression was increasing by ADAM17 and counteracted by ADAM17 siRNA, TAPI-2, and LY294002 in U87 cells. ADAM17 activated, whereas ADAM17 siRNA, TAPI-2, and LY294002 deactivated the EGFR-PI3K-AKT signal pathway, which correlated with U87 cell malignant phenotype changes.

Conclusion: This study suggests ADAM17 contributes to glioma progression through activation of the EGFR-PI3K-AKT signal pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1134. doi:10.1158/1538-7445.AM2011-1134
Abstract 1135: PPAPDC1B isoform 2, a new candidate oncogene within the 8p11-12 amplicon induces a proliferative advantage in breast cancer
Authors: Liu Gang , Yang Zeng-Quan , Ethier Stephen P.
Published: June 2012

The 8p11-12 genomic region is amplified in about 10-15% of human breast cancer and may be associated with poor prognosis. Earlier, we used genomic analysis of copy number and gene expression to perform a detailed analysis of the 8p11-12 amplicon for identifying candidate oncogenes in breast cancer. We identified phosphatidic acid phosphatase type 2 domain containing 1B (PPAPDC1B) as one of the candidate oncogenes based on statistical analysis of copy number increase and over expression.

Recently, fine-mapping of this locus, and the updated human genomic database has shown there may be more than 3 splicing variants of PPAPDC1B. We measured the expression status of all 3 individual PPAPDC1B isoforms in breast cancer cell lines by real-time quantitative RT-PCR and western blot. Interestingly, the short splicing variant /isoform2 is the dominant over-expressed form in SUM44 and SUM52 breast cancer cell lines that harbor the 8p11-12 amplicon.

The PPAPDC1B gene encodes a novel type of transmembrane phosphatidate phosphatase with an acid-PPc domain with broad lipid phosphate substrate specificity. This suggests that PPAPDC1B may play a role in cell signaling and lipid metabolism. The PPAPDC1B isoform 2 is predicted to have an inactive acid-PPc domain due to truncation of mRNA.

To address the question of whether PPAPDC1B possesses transforming properties, and to functionally characterize the different isoforms, we established lentiviral expression constructs of full-length PPAPDC1B as well as the short isoform. MCF10A cells were infected with equalized viral titer of lentiviral vectors and assayed for alterations in growth rates, colony formation and growth factor-independent proliferation. Cell proliferation assays indicated that over expression of PPAPDC1B isoform 2 enhanced cell proliferation more dramatically than full-length PPAPDC1B. Furthermore, colony formation assay showed that MCF10A cells over expressing PPAPDC1B isoform 2 formed large highly proliferative colonies compared with control cells. All of the isoforms of PPAPDC1B could induce insulin-like growth factor-independence in MCF-10A cells, but not independence of EGF. Thus, our data suggest that PPAPDC1B isoform 2 is a candidate oncogene from the 8p11 region that can induce a proliferative advantage in subset of breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1135. doi:10.1158/1538-7445.AM2011-1135
Abstract 1138: Breast cancer-derived mutations abolish Na+/H+ exchanger regulatory factor 1 inhibition of platelet-derived growth factor signaling
Authors: Cheng Shan , Li Yang , Feng Duiping , Yang Ying , Yang Longyan , Zhou Yunying , Xiong Ying , Sun Licui , Jiang Wenguo , He Junqi
Published: June 2012

Na+/H+ exchanger regulatory factor 1 (NHERF1, also known as EBP50) is a scaffold protein known to interact with a number of target proteins, including cancer-related proteins such as phosphatase and tensin homolog deleted on chromosome 10 (PTEN), neurofibromatosis 2, spleen tyrosine kinase, platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR) and β-catenin. This suggests a possible function of NHERF1 as a negative tumor regulator. Several lines of evidence have suggested that NHERF1 is involved in carcinogenesis. However, the mechanisms by which NHERF1 plays a role in breast cancer have not yet been clearly defined. Three point mutations in nherf1 (K172N, R180W, and D301V) were recently identified in breast cancer cell lines or primary breast tumors. To gain more insight into the functional properties of NHERF1, wild-type (wt) and cancer-derived nherf1 mutations were introduced into a human non-small cell lung cancer cell line, SKMES-1. NHERF1-wt overexpression suppressed the malignant phenotypic characteristics of SKMES-1 cells, including cell proliferation, migration, and invasion. Three nherf1 mutations led to complete or partial loss of NHERF1 functions by affecting the formation of PTEN/NHERF1/PDGFRβ complex, attenuating NHERF1-mediated inhibition of PDGF-induced AKT and ERK activation, and abolishing the tumor suppressor effects mediated by NHERF1-wt. These results provide the first evidence of the functional consequences and signaling properties of breast cancer-derived nherf1 mutations, and suggest that these nherf1 mutations might play a causal role in tumor development and progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1138. doi:10.1158/1538-7445.AM2011-1138
Abstract 1140: C/ebp beta represses Arf induction by tgf-beta2
Authors: Zheng Yanbin , Chu Patricia , Skapek Stephen X.
Published: June 2012

Arf is a bona fide tumor suppressor that regulates cell proliferation through p53-dependent and -independent mechanisms. Recent studies show that Arf also has physiological functions in addition to tumor repression. Our group reported that the Arf promoter is strictly controlled during development and Tgf-beta2 is required for Arf promoter activation at several sites in the developing mouse. In a cell culture-based model, Arf induction by Tgf-beta depends on Smads 2/3 and p38 MAPK. But the transcription factors influenced by these signaling pathways are not yet clear. Using a candidate-gene approach, we have identified C/ebp beta as a negative Arf regulator, modified by Tgf-beta 2. The ability of Tgf-beta2 to increase p19Arf expression in wild-type mouse embryo fibroblasts (MEFs) and Arf promoter activity in Arf lacZ/lacZ MEFs correlated with repression of C/ebp beta. Pre-incubation of MEFs with SB431542, a Tgf-beta type I receptor inhibitor, blocked C/ebp beta repression and Arf induction. Retrovirus-mediated ectopic C/ebp beta blocked Arf induction by Tgf-beta2 in Arf lacZ/lacZ and wild-type MEFs. Similarly, RNAi-driven knock-down of C/ebp beta augmented Arf promoter activation by Tgf-beta2. Chromatin immunoprecipitation revealed that C/ebp beta binding to the Arf promoter after Arf promoter is initially activated by Tgf-beta2, and the binding dropped at 48 hours, when Arf expression was significantly induced. In summary, our results indicate that C/ebp beta is modified by and contributes to Arf regulation by Tgf-beta2. In contrast, the p38 MAPK inhibitor SB203580 did not influence C/ebp beta levels but still interfered with Arf induction. We are currently exploring the importance of C/ebp beta in Arf regulation by other signals and in other cell types, and investigating its importance in vivo in the developing mouse.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1140. doi:10.1158/1538-7445.AM2011-1140
Abstract 1141: p19Arf represses Pdgfrβ-driven pericyte proliferation in vivo
Authors: Widau Ryan C. , Zelivianskaia Anna , Skapek Stephen X.
Published: June 2012

p19Arf, encoded by the mouse Arf gene, is a critical tumor suppressor with roles regulating cell cycle arrest in embryo development and suppressing oncogenesis. Previous studies established that p19Arf controls pericyte-like cells in the hyaloid vascular system (HVS) in the primary vitreous of the developing eye. Arf-deficient mice have hyperplasic expansion of pericytes in the vitreous, a developmental defect driven by excessive Pdgfrβ signaling. We have used complementary studies of cultured cells and mouse embryos to further explore the functional interactions between p19Arf and Pdgfrβ. To address whether Pdgfrβ acts as a mitogenic or a survival factor in the Arf-deficient mouse eye, we quantified cell proliferation and apoptosis at 13.5 days post coitum (dpc) in ArfGFP/GFP embryos that had or lacked Pdgfrβ. Cell proliferation, measured by BrdU incorporation, was increased by two-fold in the vitreous in the presence of Pdgfrβ. The presence or absence of Pdgfrβ did not change the small number of apoptotic cells in the vitreous of the eye. To explore mechanisms by which Arf expression impeded Pdgfrβ signaling, we used the 10T1/2 pericyte-like cell culture model to show that retroviral expression of p19Arf significantly repressed Pdgfrβ protein. Conversely, shRNA-mediated knockdown of p19Arf enhanced Pdgfrβ in serial-passaged mouse embryo fibroblasts (MEFs). In vivo measurements of Arf mRNA extracted from vitreous samples isolated by laser capture microdissection (LCM) from mouse embryos at 13.5 dpc demonstrated increased Pdgfrβ mRNA in Arf-deficient embryos as compared to wild-type. To confirm that p19Arf repressed Pdgfrβ transcription, we demonstrated that both mature and primary Pdgfrβ RNA transcripts were repressed in 10T1/2 cells. Co-transfection of p19Arf with a luciferase reporter plasmid driven by the Pdgfrβ promoter demonstrated that a 1.6kb segment of DNA conferred Arf-dependent regulation. Chromatin immunoprecipitation (ChIP) showed that p19Arf reduced RNA Pol II binding to the proximal Pdgfrβ promoter. Overall our data show that, in the absence of Arf, Pdgfrβ drives excess proliferation in cells that would normally express Arf in the developing eye. p19Arf acts to repress Pdgfrβ transcription, in part by blocking RNA Pol II binding to the proximal Pdgfrβ promoter. We are currently using additional ChIP assays to identify how the presence or absence of Arf influences the binding of several transcription factors known or suspected of regulating Pdgfrβ expression, and we are exploring the importance of the Arf-Pdgfrβ pathway deregulation in cancer models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1141. doi:10.1158/1538-7445.AM2011-1141
Abstract 1158: Multiple molecular targeting effects by miR-7 in EGFR tyrosine kinase inhibitor-resistant lung cancer xenograft models
Authors: Rai Kammei , Takigawa Nagio , Ito Sachio , Kiura Katsuyuki , Yasuda Tateji , Shimizu Kenji , Tanimoto Mitsune
Published: June 2012

Background and Purpose: Since T790M secondary mutation in exon 20 restores the affinity of ATP to the ATP binding site of mutated EGFR, the oncogene addiction by EGFR pathway is presumed to sustain in the EGFR tyrosine kinase inhibitor (TKI)-resistant cells harboring T790M. MicroRNA-7 (miR-7) was reported to regulate signaling pathway by targeting EGFR, IRS-1 and RAF1. We previously showed that miR-7 had potential tumor suppressor effect by binding 3′-untranslated region of EGFR mRNA in vitro as predicted in silico, which could be observed in xenograft model using gefitinib-resistant lung cancer cell line (RPC-9) carrying a secondary mutation T790M from EGFR mutated (delE746-A750 in exon 19) PC-9 cell line (#4041, Annual AACR meeting, 2010). We explored the efficacy and further mechanisms in other lung cancer cell lines and xenograft models.

Material and Methods: H3255 harboring L858R and H1975 carrying both L858R mutation in exon 21 and T790M mutation in exon 20 were used as EGFR addicted cell lines, while A549 was used as the cell line driven by not EGFR but KRAS. Each anti-proliferation effect was evaluated at 60 hours after transfection of miR-7 by the cell count assay. As EGFR-TKI resistant xenograft models, we selected H1975. Two million cells were injected s.c. into the backs of the athymic mice. After confirming the tumor formation of 7 mm in the diameter, we injected 3μg of control siRNA expressing plasmid and miR-7 expressing plasmid weekly into the each tumor by using optimized cationic liposome. Protein expression was determined by Western blotting.

Results: Significant growth inhibition by miR-7 was observed in the H3255 cells (p = 0.006) and H1975 cells (p = 0.004), while inhibition in A549 cells was likely to be moderate (p =0.07). Total EGFR was decreased and phosphorylation of AKT was suppressed in all cell lines by the treatment, moreover, total IRS-1 and total RAF-1 were also suppressed. Marked anti-proliferative effect in H1975 xenograft model was observed after treatment. The tumor sizes between the control mice and the treated mice were significantly different on day 19 (714% vs 154%, p=0.02). Total EGFR, IRS-1 and RAF-1 were suppressed in the tumors of H1975 and RPC-9 one day after injection of miR-7.

Conclusion: MiR-7 by non-viral weekly delivery in two typical xenograft models of EGFR-TKI resistant cell lines was effective through downregulating EGFR, IRS-1, and RAF-1. The treatment with miR-7 expressing plasmid may be promising for the EGFR-TKI-resistant lung cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1158. doi:10.1158/1538-7445.AM2011-1158
Abstract 116: Methylcytosine binding domain protein 2(MBD2) dependent proliferation and survival of breast cancer cells
Authors: Mian Omar Y. , Wang Shou Z. , Zhu Sheng Z. , Bear Harry D. , Ginder Gordon D.
Published: June 2012

Methyl Cytosine Binding Domain Protein 2 (MBD2) functions as an interpreter of DNA methylation by binding promoter sequences with high methylated CpG content and recruiting a Nuclear Remodeling and Deacetylase (NuRD) co-repressor complex. MBD2 has been shown to bind to and mediate repression of methylated tumor suppressor genes in cancer cells where re-patterning of CpG methylation and associated gene silencing is common. We have investigated the role of MBD2 in breast cancer cell growth and tumor suppressor gene expression. We show that stable shRNA mediated knockdown of MBD2 leads to significant growth suppression of cultured human mammary epithelial cancer lines, SK-BR-3 (92%); MDA-MB-231 (77%); MDA-MB-435 (94%); with the peak anti-proliferative effect occurring only after sustained, stable knockdown and lasting over 4 weeks. MBD2 knockdown also leads to markedly decreased propensity for these aggressive breast cancer cell lines to form in vivo xenograft tumors in BALB/C nu/nu mice. The growth effects of MBD2 knockdown are accompanied by de-repression of specific tumor suppressor genes including DAPK1 and KLK10. Chromatin immunoprecipitation assays and bisulfite sequencing demonstrate MBD2 binding directly to the hyper-methylated CpG-rich promoters of both DAPK1 and KLK10. Expression of an shRNA-resistant MBD2 protein results in restoration of growth and re-silencing of the MBD2 dependent tumor suppressor genes. Our data suggest that uncoupling CpG-methylation from repressive chromatin remodeling and histone modifications by removing MBD2 is sufficient to initiate and maintain tumor suppressor gene transcription and suppress neoplastic cell growth. These results demonstrate a role for MBD2 in aggressive breast cancer cell growth and provide support for the prospect of targeting MBD2 therapeutically.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 116. doi:10.1158/1538-7445.AM2011-116
Abstract 1167: Fixation does not affect the integrity of microRNAs in archival colorectal cancer specimens over a period of 20 years
Authors: Bovell Liselle C. , Shanmugam ChandraKumar , Katkoori Venkat , Salih Chura , Grizzle William E. , Manne Upender
Published: June 2012

Background: MicroRNAs (miRNAs) have potential prognostic and therapeutic value in human malignancies, including colorectal cancer (CRC). Many human tumor tissues are available as formalin-fixed paraffin-embedded (FFPE) specimens, which are an excellent source for biomarker discovery. However, the stability of miRNAs and the reliability of measurements of miRNA expression in archival samples stored for long periods of time (> 20 years) are not known. Therefore, we evaluated the stability and expression levels of miRNAs in FFPE CRC specimens and compared their expression levels to those in CRC cell lines.

Methods: A panel of seven miRNAs (RNU6B, miR-20a, miR-21, miR-106a, miR-181b, miR-203, and miR-324-5p) in 219 CRC archival tissue samples, stored for 6 to 28 years (1982 through 2004) in a dark, cool place, and 11 CRC cell lines (LoVo, HCT116, HCT116 (p53-/-), RKO, LS174T, CaCo-2, Colo205, SW480, WiDr, HCT-15, and DLD-1) were evaluated by the use of TaqMan® microRNA assays and by quantitative real-time polymerase chain reactions. In CRCs, these miRNAs, excluding RNU6B (an endogenous reference molecule), are candidate prognostic or predictive biomarkers. All assay reagents used were from Applied Biosystems Inc., (Carlsbad, CA). From each block, tissue sections, 10-microns thick, were used for extraction of miRNAs. miRNA expression was measured as threshold cycle (Ct) values. Triplicate reactions were averaged for each miRNA, and the raw Ct values were determined. The Kruskal-Wallis one-way analysis of variance was employed to analyze the miRNA expression difference among archived samples from different years. Correlation coefficient analysis was performed to compare the expression levels of miRNA in archival tissues and cell lines.

Results: In both CRC archival tissues and cell lines, the median Ct values of all miRNAs ranged from 19 to 32. All seven miRNAs were stable in CRC tissues stored for up to 28 years; their expression levels were similar across all ages of blocks. (Kruskall-Wallis P values for all 7 miRNAs ranged from 0.627 to 1.000.) There was good correlation between the levels of expression of all seven miRNAs in FFPE tissues and in the CRC cell lines (correlation coefficient R2 range, 0.784 to 0.937).

Conclusions: Since miRNAs are stable in archival CRC samples stored for over 20 years, FFPE samples can be used to evaluate miRNAs as biomarkers. These studies were supported by grants 2U54-CA118948-03, R01-CA98932, and R03-CA139629 from the NCI/NIH.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1167. doi:10.1158/1538-7445.AM2011-1167
Abstract 1170: MicroRNA gene polymorphisms in cancer susceptibility loci 5p12, 8q24 and 11q13
Authors: Kim Hye Kyung , Chanock Stephen J. , Fang Jun , Collins Irene , Amundadottir Laufey
Published: June 2012

Using Genome Wide Association Studies (GWAS), we and others have previously identified and replicated genetic variations in chromosome 5p12, 8q24 and 11q13. Interestingly, the majority of risk-associated variations map to non-coding regions of the genome and thus, the molecular mechanism of single nucleotide polymorphisms (SNPs) underlying increased cancer risk remains unknown. To assess whether these SNPs could affect the biogenesis and function of non-coding RNA species, such as microRNAs (miRNA), here we assessed miRNA gene polymorphisms in these cancer susceptibility loci. We catalogued two miRNA genes in 5p12, sixteen miRNA genes in 8q24, and seven miRNA genes in 11q13. We re-sequenced the pri- and pre-miRNA genomic sequences in these loci using HapMap DNA samples. As a result, in 8q24 miRNA genes, we identified three new SNPs and confirmed two previously reported SNPs. In 11q13, we identified four genetic variations within different miRNA genes. However, linkage disequilibrium plot analysis of both loci didn't show any statistically significant association of any of these SNPs with their corresponding GWAS signals. Thus, we consider it unlikely that miRNA in these regions are direct downstream effectors of cancer-associated genetic variations. Nevertheless, because these risk loci are prone to genomic instability, we assessed whether the SNPs could affect miRNA gene expression since indirect associations with increased cancer susceptibility can't be excluded. To address this question, we cloned pre-miRNA genes from these loci, with or without genetic variations, into miRNA expression vectors and determined their relative expression in transfected 293T cells. We tested for an 8q24-encoded miRNA (miR-1206) or an 11q13 encoded-miRNA (miR-612) expression using miRNA TaqMan assays, and we found that the G allele polymorphism (rs2114358) in miR-1206 significantly downregulated miRNA expression levels compared to the reference A allele. On the other hand, an rs550894 (C/A) in miR-612 significantly upregulated miRNA expression levels compared to that of the reference C allele. These data indicate that the miRNA SNPs in these loci have indeed functional consequences that lead to dysregulation of miRNA biogenesis. Whether such changes in miRNA expression could also result in increased cancer risk needs to be assessed in future studies by identifying the downstream target genes of these miRNAs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1170. doi:10.1158/1538-7445.AM2011-1170
Abstract 1186: Identification of p53 regulatory microRNAs
Authors: Zhang Xin , Dobbelstein Matthias
Published: June 2012

Purpose: microRNAs (miRNAs) play very important roles in gene regulation, by interfering with target mRNA stability and/or translation. A number of specific miRNAs have been identified to function as oncogenes or tumor suppressors. We originally found that miR-192/-215 constitute part of the p53 tumor suppressor network and can act as effectors and regulators of p53. In this project, we aim to identify more p53-regulatory miRNAs to gain deeper insight into p53 regulation.

Experiments: We developed an immunofluorescence-based screen to identify p53-regulatory miRNAs. Briefly, U2OS cells, an osteosarcoma cell line with wild type p53, were transfected with a collection of miRNA expression plasmids. The cellular levels of p53 were then evaluated by subsequent immunofluorescence analysis via high content BD pathway.

Results: From our screen, we identified the known p53-regulatory miRNA miR-125b (Le et al., 2009), that can reduce the levels of p53, which in turn validated our screen method. Several other candidates identified from this screen are undergoing study in the context of the p53 tumor suppressor pathway. These miRNAs might be novel therapeutic targets for diagnosis and treatment of human cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1186. doi:10.1158/1538-7445.AM2011-1186
Abstract 119: MiR-1 downregulation plays a critical role in thyroid cell proliferation
Authors: Leone Vincenza , D'angelo Daniela , Pallante Pierlorenzo , Ferraro Angelo
Published: June 2012

MiR-1 downregulation plays a critical role in thyroid cell proliferation

V. Leone, D. D'Angelo, PL. Pallante, A. Ferraro.

Istituto di Endocrinologia ed Oncologia Sperimentale del CNR c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Facoltà di Medicina e Chirurgia di Napoli, Università degli Studi di Napoli “Federico II”, Naples, Italy.

MicroRNAs have emerged as an important class of short endogenous RNAs that act as post-transcriptional regulators of gene expression, and their expression is constantly deregulated in human cancer.

MiR-1 has been found downregulated in lung, colon and prostate cancer. The aim of our work has been to investigate the possible role of miR-1 in thyroid carcinogenesis. Therefore, we have analysed miR-1 expression in a panel of thyroid neoplasias including benign and malignant lesions. Our results show that miR-1 expression is drastically downregulated in thyroid adenomas and carcinomas in comparison with normal thyroid tissue. Interestingly, miR-1 downregulation was also found in thyroid hyperproliferative non neoplastic lesions such as goiters. These results suggested a general role of miR-1 downregulation in the control of thyroid cell proliferation. This suggestion was then confirmed by the drastic decrease in miR-1 expression following exposure of rat thyroid cells to thyrotropin (TSH), the physiological thyroid stimulator. Searching for miR-1 targets, we identified CREB1 transcriptional factor, that is the main effector of the TSH-induced thyroid cell growth, as miR-1 target, suggesting the presence of a regulatory synergistic loop including TSH, CREB1 and the miR-1. Moreover, we have demonstrated that also the CXCR4 protein and its ligand SDF1α, that are involved in cell migration and are constantly overexpressed in human thyroid carcinoma, are targets of miR-1. Functional studies demonstrate that miR-1 is able to inhibit thyroid carcinoma cell proliferation and migration validating a critical role of miR-1 downregulation in thyroid carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 119. doi:10.1158/1538-7445.AM2011-119
Abstract 1190: Integration of mRNA and miRNA signatures induced by BRCA1 gene
Authors: Tanic Miljana , Zajac Magdalena , Gomez-Lopez Gonzalo , Benitez Javier , Martinez-Delgado Beatriz
Published: June 2012

During the past years it has been well established that microRNAs (miRNAs) modulate the activity of mRNAs. Microarray-based techniques are being useful to obtain miRNA and gene expression signatures associated to different samples. The aim of this study is to integrate data from both types of microarray experiments to obtain significant relationships between miRNA profile and gene expression signature associated to the absence or presence of BRCA1 gene in a cellular system. Whole genome transcriptional mRNA profiling and global miRNA expression profiling was performed on a BRCA1 deficient, HCC1937 breast cancer cell line, and the isogenic HCC1937 expressing BRCA1. We have related each differentially expressed miRNA and their miRanda predicted transcripts targets obtained from miRBase and TargetScan, to find consistent pairs of downregulated targets for upregulated miRNAs and viceversa, which were non randomly associated (Fisher, FDR<0.05).

In our study we found over 8000 differentially expressed genes and 9 differentially expressed miRNAs between HCC1937 and HCC1937/BRCA1 cells (FDR p<0.05). Integration of these data revealed significant interactions between three differentially upregulated microRNAs and their downregulated targets. Moreover, we have evaluated the role of these experimentally consistent pairs at the biological pathways level (Ingenuity Pathway Analysis). We identified potential signaling pathways significantly repressed associated with miRNA upregulation in BRCA1-expressing HCC1937 cells, which included MAPK or NFκB pathway. Down regulation of these pathways was experimentally validated by luciferase assays.

Integration analysis between miRNA-mRNA data allows us to define genes and pathways controlled by differentially expressed miRNA upon BRCA1 expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1190. doi:10.1158/1538-7445.AM2011-1190
Abstract 1196: FZD7 plays a critical role in triple negative breast cancer proliferation
Authors: Yang Lixin , Wu Xiwei , Deng Xutao , Lau Sean , Somlo George , Yen Yun
Published: June 2012

Breast cancer is genetically and clinically heterogeneous. Triple negative cancer is a subtype of breast cancer, most of which is associated with poor outcome and the lack of benefit from target therapy. A pathway analysis in a microarray study was performed using triple negative breast tumor cells compared with non-triple negative breast tumor cells. Overexpression among several Wnt pathway genes, such as FZD7, LRP6 and TCF7 has been observed in triple negative breast tumors. To validate their function, inhibition of FZD7 using FZD7-shRNA was carried out. Noticeably decreased cell proliferation, suppressed invasiveness and colony formation in triple negative MDA-MB-231 and BT-20 cells were observed. Mechanism study indicated that these effects occurred through silencing the canonical Wnt signaling pathway, as evidenced by loss of nuclear accumulation of β-catenin and decreased transcriptional activity of TCF7. Our finding suggests that FZD7 involved canonical Wnt signaling pathway is essential for tumorigenesis of triple negative breast tumor. Thus, FZD7 may be a biomarker and a potential therapeutic target for triple negative breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1196. doi:10.1158/1538-7445.AM2011-1196
Abstract 1199: Implications of protein kinase C (PKC)α and PKCΔ on murine mammary tumor growth and metastatic dissemination; effect of retinoids
Authors: Berardi Damian E. , Bessone María Inés Díaz , Campodónico Paola B. , Motter Andrea , Bal de Kier Joffé Elisa D. , Urtreger Alejandro J. , Todaro Laura B.
Published: June 2012

The retinoid system exerts some of its effects on cell differentiation and malignant phenotype reversion through the interaction of its receptors (RAR) with different PKC isoforms.

In this work, using a murine mammary tumor cell line (LM3), we studied the interaction between RARs and PKC in vitro as well as whether the overexpression of α and Δ PKC isoforms could influence the effect of retinoids on cell proliferation both in vivo and in vitro.

Western blot assays showed that the pharmacological inhibition of PKCΔ (Rottlerin 5μM, 24h) impaired all trans retinoic acid (ATRA)-induced RARα1 translocation to the nucleus while the pharmacological inhibition of PKCα (Gö6976 5μM, 24h) did not affect this translocation. Moreover, immunoprecipitation assays showed that only PKCΔ co-immunoprecipitated with RARα1 after ATRA treatment, suggesting a physical interaction between both molecules. However, both PKC isoforms seemed to be necessary for RARs activation as determined by a gene reporter assay.

Next, by a stable transfection procedure, we overexpressed PKCα and PKCΔ in LM3 cell line. In vitro assays showed that LM3-PKCα cells presented the highest proliferative rate and were highly responsive to ATRA treatment reducing their growth capability. These genetically modified lines were also inoculated orthotopically into the mammary glands of female BALB/c mice. Ten days later, when tumors were palpable, mice were implanted with ATRA pellets (10 mg/mouse). Tumors overexpressing PKCα presented a higher growth rate and were more metastatic than control vector-transfected LM3 cells, but showed a significant response to ATRA treatment, impairing primary tumor growth and reducing lung metastases number [Md (range) 55 (20-75) vs. 16 (0-27) in non-treated and ATRA-treated LM3-PKCα cells respectively (P<0.05 Mann-Whitney test)]. Both control and PKCΔ overexpressing tumors were unresponsive to retinoid treatment.We can conclude that, while PKCα overexpressing cells were more aggressive than control ones, they showed an important response to retinoid treatment both in vivo and in vitro, indicating that a retinoid therapy could be successful in human mammary tumors with high PKCα levels. On the other hand, while we found an in vitro clear interaction between PKCΔ and RARα1, the overexpression of PKCΔ did not to modulate their response to the retinoid therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1199. doi:10.1158/1538-7445.AM2011-1199
Abstract 1207: Molecular determinants of CXCR4 trafficking dictate the metastatic potential in breast cancer
Authors: Fruhwirth Gilbert O. , Brock Adrian , Keppler Melanie , Khan Abid , Giamperi Silvia , Sahai Erik , Gillet Cheryl , Archibald Steve J. , Ameer-Beg Simon , Ng Tony
Published: June 2012

The G-protein-coupled CXC chemokine receptor 4 (CXCR4) was shown to be a major determinant in organ-specific metastasis for various cancers. The C-terminus of the receptor contains phosphorylation/ubiquitination sites and residues that interact with adaptor proteins which couple to the endocytosis machinery. Post-endocytic events that lead to a decision between degradation and recycling are hitherto poorly understood.

We used fluorescently tagged receptor molecules stably expressed in breast cancer cells to establish that mutations in the protein kinase C (PKC) site of CXCR4 affect not only the cellular equilibrium between ESCRT-mediated degradation and recycling pathways but also the invasive properties. Importantly, the obtained in vitro results excellently translate to a murine model of breast cancer, in which we found that mutation of the PKC motif influences the metastatic potential. Furthermore, by using fluorescence lifetime imaging (FLIM) to measure direct protein-protein interactions via Förster resonance energy transfer (FRET) we show for the first time that ligand-dependent dimerization of CXCR4 receptors is a crucial mechanistic step in the above processes.

Our findings reveal a new mechanism whereby the penetrance of a mutated receptor that exhibits resistance to its desensitization and confers an enhanced cancer migratory phenotype, is reduced through the dimerization with its wildtype counterpart.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1207. doi:10.1158/1538-7445.AM2011-1207