bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3198: The CEE-Selector Assay: A tool for the identification of rare allele variants
Authors: Alexiadis Vassilios , Watanaskul Tim , Kosco Karena , Arnold Lyle
Published: August 2012

Molecular assays for the identification of rare allele occurrences are important tools for proper cancer classification and treatment. A prime example is the T790M mutation in EGFR which leads to resistance to the tyrosine kinase inhibitors gefitinib (Iressa®) and erlotinib (Tarceva®) used in the treatment of non-small cell lung cancer (NSCLC). Identification of the T790 mutation in cancer-shed particles in blood (either as whole cells or subcellular vesicles) calls out the need for an alternative cancer treatment. We have developed a highly sensitive PCR-based assay which allows the identification of the T790M mutation in blood plasma (either when present in mRNA or genomic DNA). The assay combines Real-Time PCR as well as melt curve analysis of the mutant PCR product and is followed by sequencing to verify the presence of the mutation. The Selector Assay is based on a wild-type specific PCR blocker and allows the mutant template to be amplified in a high background of wild-type template. A few copies of T790M mutant can be detected in greater than a 1000-fold excess of wild-type. Data using the Selector Assay with clinical lung cancer samples showing the identification of T790M in genomic DNA as well as mRNA in blood plasma and in CTCs will be presented. The Selector Assay can be applied to other mutations relevant to cancer and is a valuable tool for clinical diagnostics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3198. doi:1538-7445.AM2012-3198
Abstract 3199: Miniaturized, multiplexed, highly sensitive protein NanoArray assay for measuring biomarkers in dried blood spot (DBS) samples
Authors: Dixon Douglas B. , Ivanov Ivan D. , Gubbins Earl J. , Harris Richard R. , Coen Michael L. , Helfrich Rosalind , Jamil Haris
Published: August 2012

As the utility of new biomarkers for diagnosis and treatment of disease become better understood, physicians and researchers are faced with increasingly critical decisions regarding how to best distribute their valuable patient samples. While simply taking more blood would seem a reasonable solution, a recent publication in the Archives of Internal Medicine (Salisbury, et.al., Arch Intern Med. 2011;171(18):1646-1653) addresses the very real problem of hospital-acquired anemia (HAA) brought on by blood loss resulting from blood drawn for clinical assays. Also in many resource constrained countries, the lack of adequate infrastructure such as hospitals, laboratories, transportation systems as well as qualified personnel pose major obstacles for patient access to clinical testing. This has revived interest in collecting dried blood spots (DBS) for clinical testing. DBS samples provide a number of benefits when compared with drawing venous blood: they require minimal blood volume (usually 50µl per spot), they can easily be collected from infants and children, and they can be used to collect blood samples where collection and storage of serum or plasma is not feasible. However for DBS samples to be a viable solution for clinical assay, the assay method must be sensitive and yet require very small sample volumes. NanoInk, Inc. has developed a highly sensitive assay based on dip-pen nanolithography (DPN™) using the NanoArrayer3000™. Utilizing a 48 subarray slide platform printed with a nano-scale antibody array we have created a miniaturized multiplexed sandwich ELISA with sensitivities as low as the 100's of femtograms/ml while using sample volumes that range from 2-4µl/well. Using our proprietary elution protocol, we are able to elute biomarkers from a standard 3.2mm DBS card punch sufficient to provide triplicate assay values while still keeping the samples concentrated enough to measure lower concentration biomarkers. Our NanoInk DBS cytokine assay has been validated using eluted blood standards and QC samples and shows good well-to-well and slide-to-slide variability. NanoInk's multiplexed assay format can also be customized to include a wide array of biomarkers. This multiplex capability, in combination with our proprietary sample and standard elution protocol, now allows clinicians to accurately measure the concentration of multiple biomarkers using convenient small-volume DBS samples. The results of an ongoing study for measuring biomarker levels in patient samples using our 5-cytokine DBS assay will be presented.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3199. doi:1538-7445.AM2012-3199
Abstract 32: P190B RhoGAP overexpression drives mammary epithelial cell proliferation by altering stromal-epithelial interactions in the developing mammary gland
Authors: Gillette Melissa , McHenry Peter , Blumenthaler Alisa , Chang Peggy , Vargo-Gogola Tracy
Published: August 2012

Breast cancer development and progression is enabled by alterations in the surrounding stroma originating from stromal interactions with cancerous epithelium. Rho GTPases and their regulators have been implicated as important mediators of stromal-epithelial interactions in mammary epithelial cells (MECs), and alterations in the microenvironment profoundly affect MEC morphogenesis in a Rho GTPase-dependent manner. Previously, we demonstrated that p190B Rho GTPase activating protein (RhoGAP), a major inhibitor of Rho GTPases, has pro-tumorigenic functions during MMTV-Neu induced mammary tumorigenesis. We hypothesized that p190B overexpression in the epithelium promotes mammary tumor development by altering stromal-epithelial interactions to drive MEC proliferation and disrupt tissue architecture. To investigate this hypothesis, we examined the effects of p190B overexpression in the developing mammary gland (MG) using Tet-regulatable p190B overexpressing mice. Interestingly, overexpression of p190B in the mammary epithelium during postnatal MG development causes hyperbranching and disorganization of the ductal tree. Using a combination of in vivo morphometric and immunohistochemical analyses and in vitro stromal-epithelial co-cultures, we have demonstrated that p190B overexpression in MECs activates fibroblasts in the adjacent microenvironment leading to increased extracellular matrix (ECM) deposition, elevated expression of the collagen crosslinking enzyme lysyl oxidase, and increased expression of potent regulators of branching morphogenesis, including FGF and TGFβ growth factors. These stromal alterations correlated with increased proliferation of p190B overexpressing MECs in vivo. Taken together, these data suggest that p190B overexpression may contribute to mammary tumorigenesis by creating a pro-tumorigenic stromal environment leading to increased MEC proliferation and altered tissue architecture.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 32. doi:1538-7445.AM2012-32
Abstract 320: Investigating the patterns of nuclear factor-kappa B activity in the host-tumor microenvironment during ovarian cancer progression
Authors: Wilson Andrew J. , Lee Hye-Jeong , Barham Whitney , Chen Lianyi , Onishko Halina , Khabele Dineo , Yull Fiona
Published: August 2012

Most women diagnosed with ovarian cancer initially present with metastatic disease characterized by peritoneal implants and ascites. Activation of inflammatory processes mediated by nuclear factor-kappa B (NF-κB) is thought to be critical to the distinct clinical pattern of spread of ovarian tumors. However, the relative contribution of NF-κB activity in tumor cells and host inflammatory cells to ovarian cancer progression remains unknown. Therefore, our goal was to develop and characterize model systems to investigate NF-κB activity in these cell populations during ovarian tumorigenesis. To study the host NF-κB response, ID8 mouse ovarian cancer cells were injected intra-peritoneally into C57BL/6 mice carrying an NF-κB-dependent green fluorescent protein (GFP)/luciferase fusion transgene (NGL). In a reciprocal approach, ID8 cells stably expressing the NGL reporter (ID8-NGL) were injected into wild-type C57BL/6 mice to investigate NF-κB activation in the developing tumor. Non-invasive imaging of the mice was performed by bioluminescence imaging (BLI) of the NGL reporter and by positron emission tomography (PET-CT) imaging of abdominal tumor burden. Preliminary experiments confirmed these models were highly reproducible, with peritoneal-wide tumor dissemination accompanied by the onset of late-stage, irreversible ascites. Mice injected with ID8-NGL cells showed greater than 100-fold increase in NF-κB reporter activity during tumor progression. There was a modest overall decrease in abdominal NF-κB activity over time in ID8-injected NGL mice, although the possibility of “masking” effects of the ascites fluid on bioluminescence, and NF-κB activity in specific host cell populations, need to be elaborated. These models will allow us to define the patterns of NF-κB activity in the host-tumor microenvironment during ovarian cancer progression, and will provide a powerful platform for future preclinical investigation of novel therapeutic agents targeting NF-κB in ovarian cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 320. doi:1538-7445.AM2012-320
Abstract 3202: Universal detection of protein phosphorylation based on multi-functionalized soluble nanopolymers
Authors: Iliuk Anton , Pan Li , Liu Shawn , Martinez Juan , Hall Mark , Liu Xiaoqi , Tao Andy
Published: August 2012

Protein phosphorylation, as the most common covalent modification of proteins, plays a critical role in the regulation of many cellular functions. Irregularities in phosphorylation network are a major cause of onset and progression of many diseases, most notably cancer. Consequently, detection of protein phosphorylation is essential in further understanding of cellular signaling pathways and mechanisms of cell growth and proliferation. To assist in effective phosphorylation analyses, we introduce here a novel technique based on soluble nanopolymers with excellent solubility, compact spherical shape and chemical homogeneity. Soluble nanopolymers functionalized with Ti(IV) ions have shown highly versatility and allow for highly efficient analyses of phosphorylation and signaling pathways. We devise here a novel strategy, termed pIMAGO (phospho-imaging), for effective detection of phosphoproteins on a membrane (Western Blot format) or in a 96-well plate (ELISA format). Unlike antibody-based strategies, the new method is capable of selectively binding a phosphorylated residue independent of amino acid microenvironment. It shows no preference for either of the phosphosites, and thus holds great promise in biological analyses where the site of phosphorylation is not known or its specific antibody is not available. Using this technique, the phosphorylation levels of proteins of interest under physiological conditions can be readily detected as part of a standard Western Blot or ELISA procedures without the need for radioactivity or expensive phosphosite-specific antibodies. The utility of the approach has been demonstrated using standard mixtures of proteins, by in vitro kinase and phosphatase assays, as well as with kinase inhibitor screening and enzyme profiling. In addition, multiplexing capabilities of pIMAGO are presented, a feature often unavailable during antibodies-based detection. Finally, we further confirmed that the technique is sensitive and specific enough to detect endogenous phosphorylation changes by analyzing physiological signaling in number of endogenous protein complexes isolated directly from cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3202. doi:1538-7445.AM2012-3202
Abstract 3204: Novel, rapid and sensitive technology to analyze gene expression directly from archived FFPE clinical oncology specimens without sample preparation
Authors: Kim Chang Hee , Gerstner Arpad , Abedi Majid R. , Childers John R. , Terbrueggen Robert
Published: August 2012

Formalin-Fixed Paraffin Embedded (FFPE) tissue samples are the standard for clinical oncology tissue sample archiving worldwide. It is estimated that there are approximately a billion FFPE tissue cancer samples archived in hospitals and tissue banks around the world, and many of these samples have full clinical annotation including long term follow-up data. Current methods of analyzing gene expression in these samples involve laborious and expensive sample preparation protocols and long turnaround times (2 days or longer). We have developed a novel, rapid (3 hours), sample-to-answer technology to analyze gene expression from clinical FFPE tissues without the need for RNA extraction. The technique is highly sensitive and requires only 2 mm x 5 mm x 10 microns of FFPE material scraped from a pathology slide. The gene expression data obtained directly from FFPE without sample preparation correlates highly with data obtained on RNA extracted from the same FFPE sample using conventional methods. Importantly, the gene expression data obtained from prostate and breast FFPE samples correlate highly with microarray data, and with data from matching mirrored FF (Fresh Frozen) samples. The assay can be developed for detection using Luminex instrument or Capillary Electrophoresis instrument (Caliper GX or ABI 3500).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3204. doi:1538-7445.AM2012-3204
Abstract 3205: Isolating RNA, DNA and protein from the same sample allows correlation of multiple biomarkers
Authors: Fekete Richard A. , Karberg Mike , Cheng Angie , Chapman Laura , Gonzalez Marie
Published: August 2012

DNA, RNA or proteins are routinely analyzed as markers to define the type and state of tumorigenicity. Each of these analytes provide a unique set of information in order to detect differences such as chromosomal mutations, imbalances in gene expression, and changes in protein signaling networks. Limited samples, such as biopsies and small tumors, are a common sample type, however, due to their small size the analysis of more than one of these analytes from the same sample can be challenging. Taking more than one sample (biopsy), or splitting a sample in order to isolate one of these anlaytes from each section raises questions regarding sample heterogeneity. This is particularly important where one section of a tumor may be different from another, and can even provide misleading information regarding the correlation of DNA, RNA or protein markers. Another issue raised with splitting samples is that two of the analytes are lost when the third is isolated. This can lead to the loss of important information, and the low yield can limit the number and methods used for analysis. Being able to analyze DNA, RNA and proteins from the same sample is particularly crucial during biomarker discovery and correlation. Here we describe a new, quick and easy to use method to isolate DNA, RNA and proteins from small sized tumor and normal adjacent tissue and demonstrate that not only is the entire sample maintained giving us more sample for analysis (no analyte is lost when isolating the other), but there is no concern regarding heterogeneity. Using these samples we demonstrate the correlation of DNA, mRNA, miRNA and protein using Next Generation Sequencing, qPCR, Western and ELISA and associate these with tumorigenicity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3205. doi:1538-7445.AM2012-3205
Abstract 3255: IKBKE is induced by STAT3 and tobacco carcinogen and determines chemosensitivity in non-small cell lung cancer
Authors: Guo Jianping , Kim Donghwa , Kurtyka Courtney , Chen Huihua , Wu Danwei , Mittal Aman , Cheng Jin Q.
Published: August 2012

Serine/threonine kinase IKBKE is a newly identified oncogene, however, its regulation remains elusive. Here, we provide evidence that IKBKE is a downstream target of STAT3 and that tobacco induces IKBKE expression through STAT3. Ectopic expression of constitutively active STAT3 led to increase of IKBKE mRNA and protein levels, whereas inhibition of STAT3 reduced IKBKE expression. Further, ex-pression levels of IKBKE are significantly associated with STAT3 activation and tobacco use history in NSCLC patients examined. In addition, we show induction IKBKE by two components of cigarette smoke, nicotine and the NNK. Upon exposure of nicotine or NNK, cells express high levels of IKBKE protein and mRNA, which are largely abrogated by inhibition of STAT3. Characterization of the IKBKE promoter revealed 2 STAT3 response elements that directly bind to STAT3 and respond to nicotine and NNK stimulation. Notably, knockdown of IKBKE not only selectively sensitizes NSCLC cells to chemotherapy but also abrogates the protective effects of constitutively activate STAT3 and nicotine on gemcitabine and cisplatin-induced apoptosis. These data indicate for first time that IKBKE is a direct target of the STAT3 and is induced by tobacco carcinogen through STAT3 pathway. In addition, our study also suggests that IKBKE is an important therapeutic target and could play a pivotal role in tobacco-associated lung carcinogenesis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3255. doi:1538-7445.AM2012-3255
Abstract 3257: Nodal signalling in choriocarcinoma cells
Authors: Law Jeffrey , Luca Alice , Zhang Guihua , Postovit Lynne-Marie , Bhattacharya Moshmi
Published: August 2012

Choriocarcinoma is an aggressive cancer that arises when the invasion of placental trophoblast cells into the endometrium becomes dysregulated. Trophoblast invasion is a complex phenomenon, dependent on cues that drive trophoblast invasion, as well as factors to ensure that placental invasion does not extend beyond the myometrium. Nodal, a member of the TGF-β superfamily, is expressed throughout the endometrium during the peri-implantation period and in invasive trophoblast cells. However, Nodal's exact role in placental function is unclear. Nodal signals by activating the ALK4/7, ActRIIB and Cripto-1 receptor complex and emerging evidence suggests that its signalling strength can be controlled at the level of receptor endocytosis into signaling endosomes. Two key regulators of receptor endocytosis and cell invasion are the α-arrestins, multifunctional scaffolding proteins and Ral GTPases, members of the Ras superfamily. Ral can bind to ActRIIB, and its activity can be regulated by Ras, as well as by α-arrestins, in a Ras-independent manner. We thus hypothesized that Nodal promotes trophoblast invasion via a α-arrestin/Ral-mediated pathway. The specific objectives of my study are to (i) determine the effects of Nodal on trophoblast invasion, (ii) elucidate a role for α-arrestin / Ral signaling in regulating Nodal-induced trophoblast invasion. We found that Nodal stimulates the invasion of both the non-malignant HTR-8SV/neo trophoblast and JAR choriocarcinoma cells using Matrigel-coated transwell chamber assays. Interestingly, we found that the JAR choriocarcinoma cells responded differently to Nodal in that while HTR-8SV/neo invasion returned to baseline levels, JAR choricocarcinoma cells continued to invade even at the higher concentrations of Nodal (100ng/mL & 500ng/mL). For this reason, we decided to look at potential differences in the regulation of Nodal-induced invasion between the non-malignant HTR-8SV/neo trophoblast and JAR choricoarcinoma cell lines. We found that the JAR choriocarcinoma cells differentially expressed Ral and α-arrestin proteins compared to the HTR-8SV/neo cells. Additonally, endogenous Ral and α-arrestins associate with the ALK4 and 7 receptors, and knockdown of these molecules impaired Nodal-induced invasion. Thus, our data reveal a novel function for Nodal in modulating trophoblast invasion via a α-arrestin/Ral signaling pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3257. doi:1538-7445.AM2012-3257
Abstract 3258: Targeted-deletion of Ptp4a3 suppresses murine colon tumorigenesis
Authors: Zimmerman Mark W. , Homanics Gregg E. , Lazo John S.
Published: August 2012

Protein tyrosine phosphatase 4a3 (Ptp4a3, PRL-3) is a small, prenylated, 4a-family phosphatase that is highly conserved throughout mammalian species. Elevated expression levels commonly seen in human cancers, particularly metastatic colorectal cancer, suggest that Ptp4a3 may serve as a target for novel anticancer therapeutics. Ectopic overexpression of Ptp4a3 in cell culture models often enhances the cellular properties of migration and invasion. While Ptp4a3 has historically been associated with late-stage disease and metastatic progression, we observed increases in Ptp4a3 mRNA in the colon immediately following treatment with the colon pro-carcinogen azoxymethane (AOM). In the AOM colon cancer model, wildtype C57BL/6 mice develop approximately 7-10 tumors per mouse in the distal colon. The resulting tumors exhibit an average of 4-fold upregulation of Ptp4a3 mRNA relative to normal colon epithelium, as well as positive immunohistochemical staining indicating the presence of PTP4A3 protein. To further understand this promising target, we have created the first Ptp4a3 knockout mouse model. Ptp4a3 knockout mice are viable and appear normal, although male mice appear to have a slightly reduce body mass index. Interestingly, Ptp4a3 knockout mice developed fewer colon tumors than wildtype mice with the AOM model, suggesting that Ptp4a3 may have a role in colon tumorigenesis and the cellular response to genotoxic stress.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3258. doi:1538-7445.AM2012-3258
Abstract 3259: TGFβ signaling is frequently attenuated in hepatocellular carcinomas but is retained for malignant phenotypes in some hepatocellular carcinoma cells
Authors: Mu Xiaoxin , Herzig Maryanne C. , Washburn William K. , Walter Christi A. , Halff Glenn A. , Sun Lu-Zhe
Published: August 2012

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. Its development and progression have been shown to be regulated by various cytokines including TGFβ. However, the role of TGFβ signaling in the progression of HCC remains controversial. In the current study, we examined the expression levels of TGFβ pathway components and the activation status of Smad2/3 proteins as reflected by phospho-Smad2/3 levels in human and mouse HCC tissues and human HCC cell lines. We also investigated whether abrogation of TGFβ signaling in HCC cell lines that are responsive to TGFβ affects the growth and metastasis of transplanted HCC cells in nude mice. Quantitative real-time RT-PCR and Western immune blotting analyses reveal widespread down-regulation of TGFβ signaling pathway components including Smad2/3 and TGFβ type I and II receptors in HCC tissues of patients and mice in comparison to adjacent normal tissues. Treatment of our HCC cell lines (SNU398, SNU423, HepG2, and Sk-Hep-1) with TGFβ1 revealed that the SNU398 cell line was refractory whereas the other three cell lines were sensitive to TGFβ as determined with a number of in vitro assays including Smad phosphorylation, gene transcription, and growth inhibition assays. Knockdown of TGFβ type II receptor with shRNA significantly reduced TGFβ signaling activity in the Sk-Hep-1 cell line resulting in suppressed growth of its subcutaneous tumors and reduced metastasis potential when the cells were inoculated through tail vein injection. Our results suggest that TGFβ signaling may play a suppressive role in early stage of HCC development, but a promoting role in advanced stage, which is represented by the Sk-Hep-1 cell model.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3259. doi:1538-7445.AM2012-3259
Abstract 3260: Primary cilia are lost early in prostate cancer progression
Authors: Hassounah Nadia , McDermott Kimberly , Nagle Raymond
Published: August 2012

Prostate cancer is the most prominently diagnosed cancer in men, aside from non-melanoma skin cancer, and the second leading cause of cancer related deaths in men in the U.S. Little is known about the role of primary cilia in prostate cancer progression, so our goal is to elucidate this role. Primary cilia are microtubule-based organelles present on many mammalian cell types and help the cell sense the extracellular environment. Various signaling pathways important in development involve primary cilia, such as the Hedgehog (Hh) and Wingless/Int (Wnt) pathways, and these pathways are known to be misregulated in prostate cancer. Dysfunction of primary cilia is the cause of numerous ciliopathies, such as polycystic kidney disease and Bardet-Biedl syndrome, and has been linked to cancers, like basal cell carcinoma and medulloblastoma. We hypothesize that primary cilia suppress prostate tumorigenesis, and cilia loss promotes prostate cancer by altering cell signaling pathways like Hh and canonical Wnt. To test this hypothesis, we looked in human prostate cancers at cilia, Hh and canonical Wnt signaling. Human prostate tissue from prostate cancer patients was stained for ciliary proteins (acetylated and gamma tubulin). The expressions of these proteins were analyzed using confocal microscopy and compared between adjacent normal, hyperplastic, prostatic intraepithelial neoplasia (PIN), malignant and perineural invasion areas of each patient's tissue. As a control, normal prostate tissue from bladder cancer patients who underwent prostatectomies as part of treatment was used. A marked decrease in the percentage of ciliated cells in cancer and PIN was observed compared to normal prostate tissue. Higher grade cancer (Gleason sum α7) also had fewer cilia than lower grade (Gleason sum = 6). We are currently investigating Hh and canonical Wnt signaling activity in serial sections of the tissue used to analyze primary cilia. The expressions of Beta-Catenin, a protein used to measure canonical Wnt signaling activity, and Gli1, a transcription factor used as a readout of Hh activity, are being analyzed based on expression levels and localization within the cell, and correlated to primary cilia data. Thus far, our results suggest a role of primary cilia in promoting prostate cancer progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3260. doi:1538-7445.AM2012-3260
Abstract 3261: GRP78 regulates clusterin stability, retrotranslocation, and mitochondrial localization under ER stress in prostate cancer
Authors: Li Na , Zoubeidi Amina , Beraldi Eliana , Gleave Martin E
Published: August 2012

Expression of clusterin (CLU) closely correlates with regulation of apoptosis in cancer. While endoplasmic reticulum (ER) stress-induced up-regulation and retro-translocation of cytoplasmic CLU (cCLU)(also termed as presecretory and secreted forms, psCLU and sCLU) has been linked to its anti-apoptotic properties, the mechanism mediating these processes remain undefined. Here, we show that GRP78 (Bip) associates with cCLU to facilitate its retro-translocation and redistribution to the mitochondria in human prostate cancer cells. We found that ER stress inducers, such as TG or MG132, increased expression levels of both cCLU and GRP78 proteins and led to the association of these two proteins. Furthermore, GRP78 overexpression resulted in increased cCLU but reduced sCLU levels secreted into the culture media. GRP78 stabilized cCLU protein and its hypo-glycosylated forms, in particular after paclitaxel treatment. Moreover, subcellular fractionation studies indicated GRP78 increased stress-induced cCLU retro-translocation from the ER with redistribution to the mitochondria. Live cell imaging revealed that a CLUGFP localized directly to highly mobile mitochondria. Co-expression of both GRP78 and CLU in LNCaP cells led to increased CLUGFP -mitochondria co-localization and reduced apoptosis by cooperatively stabilizing mitochondrial membrane integrity. Transient depletion of GRP78 reduced cCLU protein but not mRNA transcript levels, accompanied by cell cycle arrest at G0/G1 with down-regulation of both phospho-Rb and cyclin D1, and synergistic increases in paclitaxel-induced cell apoptosis. Taken together, our findings reveal a novel role of GRP78 in the regulation of CLU retro-translocation and its redistribution to the mitochondria, suggesting that GRP78 and CLU cooperatively promote survival during ER stress in prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3261. doi:1538-7445.AM2012-3261
Abstract 3264: Downregulation of intersectin-1s in human lung cancer may contribute to tumorigenesis
Authors: Predescu Dan , Zhang Jin , Patel Monal , Bardita Cristina , Predescu Sanda
Published: August 2012

The expression of intersectin-1s (ITSN-1s), a granzyme B (GrB) substrate and one of the trisomic genes that may provide protection against most cancers in Down's syndrome, is significantly downregulated in human lung cancer (LC) cells and tissue. To date, there is no information on the cellular and molecular factors that modulate ITSN-1s expression or whether downregulation of ITSN-1s can contribute to LC. Thus, we hypothesize that loss of full-length ITSN-1s due to proteolytic cleavage by GrB during an inflammatory response and the interplay between the signaling events triggered by the two cleavage products, are part of a complex common molecular mechanism involved in the oncogenic transformation of cells. To address this hypothesis, we used cultured LC cells and tissue, as well as a range of biochemical (Western blot, ELISA, BrdU incorporation), molecular biology (plasmid DNA transfection, qPCR), morphological (immuno-histochemistry, fluorescence microscopy) approaches, soft agar colony formation and xenograft tumor formation in nude mice to evaluate whether downregulation of ITSN-1s can contribute to lung carcinogenesis. Western blot and immuno-histochemistry of LC cells and lung tissue lysates showed significant decrease in ITSN-1s protein levels, while qPCR revealed only slight decrease, if any, in ITSN-1s mRNA. In vitro studies showed that GrB cleaves ITSN-1s and generates a cleavage fragment (GrB-EHITSN) with high proliferative potential. Lung epithelial cells expressing the GrB-EHITSN show 40% increase in BrdU incorporation, selective activation of p38MAPK and downstream Elk-1 transcription factor. These findings are consistent with a p38MAPK and Elk-1 pro-proliferative signaling pathway regulated by ITSN-1s. Activation of p38 and Elk-1 was also detected by western blot using phospho-specific antibodies, in all LC cells investigated. Expression of the two GrB cleavage fragments of ITSN-1s, individually or together, in human lung epithelial cells exerts a modulatory effect on the balance p38/Erk1/2MAPK activation. Functional studies showed that ectopic expression of ITSN-1s in the LC cells restored the p38/Erk1/2MAPK balance, significantly reduced their proliferation rate as well as the formation of colonies in soft agar and xenograft tumors in nude mice. Altogether, our results suggest that downregulation of ITSN-1s can promote the oncogenic transformation of cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3264. doi:1538-7445.AM2012-3264
Abstract 3265: PMA induces malignant characteristics in MCF-7 breast cancer cells via MAPK-pathway
Authors: Hautala Laura C. , Koistinen Riitta , Koistinen Hannu
Published: August 2012

Phorbol 12-myristate 13-acetate (PMA) is a well known tumor promoter, the effect of which is mainly mediated by protein kinase C (PKC). The effect of PMA varies depending on the cell line and growth conditions. In MCF-7 breast cancer cells PMA has been found to induce growth arrest and resistance to apoptosis. Here, we studied the effects of PMA on differentiation and malignant characteristics of MCF-7 cells. The cells were grown on Matrigel besement membrane preparation or on plastic/glass surface with and without PMA, rottlerin or bryostatin. The cell proliferation assay was carried out using the cells grown on 96-well plates. Wound healing test, on glass surface, was used to detect the differences in cell migration. Activation of MAPK-pathway proteins was studied with Phospho-kinase array. When grown on Matrigel, the addition of PMA to cell culture medium increased the motility of the cells and induced dramatic morphological changes, leading to the formation of web-like multicellular structures. These changes were not observed when the cells were grown on plastic or glass surface. However, PMA caused a two-fold increase in the migration of the cells on glass surface. Furthermore, the PMA treated cells had lower proliferation rate than the untreated cells. Preincubation of the cells with PKC-inhibitors rottlerin or bryostatin abolished the effects of PMA, suggesting that these effects are mediated by PKC. PMA also induced several changes in the phosphorylation of different proteins involved in MAPK-pathway. Our results confirm the previous findings showing that PMA induces malignant characteristics in MCF-7 breast cancer cells. Morphological differentiation was only seen when the cells were grown on basement membrane preparation, recapitulating the physiological growth environment of the cells better than the artificial plastic surface. Furthermore, our results indicate that the effects of PMA on MCF-7 cells are mediated by MAPK-pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3265. doi:1538-7445.AM2012-3265
Abstract 3269: Basal VEGF protein expression in skeletal muscle, adipose tissue and mammary tumors of PyMT mice
Authors: Vona-Davis Linda , Audet Gerald , McLaughlin Sarah , Olfert Mark
Published: August 2012

Obesity is associated with the overall risk of certain cancers, including breast cancer. The prevailing view is that metabolic alterations associated with obesity result in the production of excess vascular endothelial growth factor (VEGF) thereby establishing a local and systemic environment that favors the development and progression of breast cancer. We need to understand how VEGF from adipose is integrated with mammary tumor growth and vascular activity to modulate tumor progression. The MMTV-PyMT mouse is a clinically-relevant model for studying breast cancer progression as mammary tumors grow spontaneously over time; however, little is known about the distribution of VEGF between various organs. We sought to compare the basal VEGF protein expression in skeletal muscle, adipose tissue and mammary tumors from MMTV-PyMT mice and compared these levels against background nontransgenic FVB/N mice. Normal expression of VEGF protein was 69.4 ± 2.1 pg/mL in skeletal muscle and 59.0 ± 7.3 pg/mL in adipose tissue of wild-type (FVB background) control mice. The average expression level of VEGF protein in mammary tumors of MMTV-PyMT mice was 61.8 ± 31.3 pg/mL. Adipose tissue of MMTV-PyMT exhibited 324% greater (191.2 ± 37.6 pg/mL, p<0.01) VEGF protein expression compared to adipose tissue of control FVB mice, and 309% greater (p<0.05) VEGF protein compared to tumor VEGF expression. These data imply that stromal expression of VEGF may be a critical determinant regulating the angiogenic phenotype in breast cancer, and further suggests that cross-talk between the stromal environment and tumor is likely to be essential in promoting tumor growth and progression. NIH 5T32-HL090610, AHA 10BGIA3630002, ACS 116837IRG0906101

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3269. doi:1538-7445.AM2012-3269
Abstract 3270: The effect of soy exposure in different ages on the mammary tumor development of the MMTV-ErbB-2 transgenic mice
Authors: Feng Xiaoshan , Hou Jianfeng , Gao Shegan
Published: August 2012

Objective:To investigate the effect of different soy exposure timings on the mammary tumor development using MMTV-erbB-2 transgenic mice. Furthermore, the underlying mechanisms were also examined. Methods:120 MMTV-erbB-2 transgenic female mice were randomly divided into four groups:control group, early exposure group, late exposure group and lifelong exposure group. The control group mice were treated with soy free diet from week 3 to week 60, the experiment groups mice were treated with soy diet between week 3 to week 12, week 20 to week 60, week 3 to week 60, respectively. Then, the body weight change of each mice, the latency period, and the growth speed of mammary tumor were recorded. Furthermore, the mammary gland morphology was examined by whole mount at 5 weeks. In order to understand the underlying mechanisms of the mammary tumor development, the signal pathways of c-Fos, erbB2 and p-ERK were also examined in breast cancer tissue using immunohistochemistry. Results:We found that the weight in the four groups had no significant difference. Compared with the control group, the TEB numbers and the growth speed of tumors in the early exposure group and the lifelong exposure group were significantly decreased and the tumor latency periods were significantly prolonged (P<0.05). Furthermore, immunohistochemistry showed that the mice in the early exposure group and the lifelong exposure group displayed decreased expression of c-Fos and p-ERK (P<0.05). The expression of erbB-2 protein among the different groups was not statistically significant (P>0.05). Conclusion: Early or lifelong soy exposure affects the morphological development of mammary gland of MMTV-erbB-2 transgenic mice. Soy may display a preventive effect on the initiation of breast cancer. These obersveration may serve as a guide for soy use in the early prevention of the breast cancer. The c-Fos and and p-ERK may be involved in the development of mammary tumor in the MMTV-erbB-2 transgenic mice. The underlying mechanism warrants further studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3270. doi:1538-7445.AM2012-3270
Abstract 3271: Targeting fibroblast growth factor receptor 4 decreases prostate cancer growth and metastasis
Authors: Feng Shu , Yu Wendong , Cai Yi , Wang Jianghua , Ittmann Michael M.
Published: August 2012

FGFR4 up-regulation occurs in advanced prostate cancers. Multiple FGFs have been found upregulated in prostate cancers, including FGF1, FGF2, FGF6, FGF8 and FGF17, all of which can activate FGFR4. Furthermore, a SNP changing the sense codon 388 from glycine to arginine in the transmembrane domain of the human FGFR4 gene has been linked to prostate cancer initiation, progression and poor prognosis. In this study, we determined whether downregulation of FGFR4 expression using small hairpin RNA (shRNA) against FGFR4 inhibits prostate cancer progression in vitro and in vivo. We used two well-characterized prostate adenocarcinoma cell lines, PC3 and LNCaP to derive prostate cancer cells stably expressing a shRNA targeting FGFR4 (PC3-sh-FGFR4 and LNCaP-sh-FGFR4) by using lentivirus vector infection. Real-time quantitative RT-PCR shows that FGFR4 mRNA expression was decreased by 87% (PC3-sh-FGFR4) and 78% (LNCaP-sh-FGFR4) compared to scramble control shRNA cells PC3-shV and LNCaP-shV, respectively. In vitro analysis showed that the targeted knockdown of FGFR4 gene in PC3 and LNCaP cells with sh-FGFR4 resulted in significantly inhibited cell proliferation and invasion. The reduction of Akt phosphorylation on both Ser473 and Thr-308 sites detected by western blot suggests that FGFR4 signaling is implicated in Akt activation. The upregulated expression of Caspase 8 and activated Caspase 3 in FGFR4 knockdown cells measured with immunoblot indicates that FGFR4 signaling inhibits prostate cancer cell apoptosis. The prostate cancer orthotopic xenograft models established with PC3-sh-FGFR4 and scramble shRNA control PC3 cells reveal that FGFR-4 knockdown significantly inhibited tumorigenicity in this model (P=0.01). In mice with pathologically confirmed primary tumors, primary tumor weight was decreased 63% in PC3-shFRGR-4 tumors (p<0.05) and the proportion of mice with lymph node metastasis was decreased from 93.3% to 50% (p<0.01). The Ki67 immunohistochemistry showed a significant decrease of Ki67 positive cell percentage in PC3-sh-FGFR4 xenograft tumors (19.85%) compared to control group (35.93%, p<0.001) indicating that the reduced primary tumor size was associated with the decreased cell proliferation. The orthotopic xenograft tumors produced with LNCaP-shFGFR4 showed 67.5% decreased weight compared to the tumors of control LNCaP-shV cells (p=0.0157). Our data indicated that FGFR4 can be a promising target in prostate cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3271. doi:1538-7445.AM2012-3271
Abstract 328: The mitotic kinase Aurora-A promotes breast cancer metastases by inducing a CD24low/- basal-like stem cell phenotype
Authors: D'Assoro Antonino B.
Published: August 2012

In this study we demonstrate that gain of HER-2/Neu expression in human breast cancer xenografts harboring constitutive active Raf-1 induces Aurora-A over-expression that ultimately drives the transition from a luminal to a highly invasive basal-like phenotype. Over-expression of Aurora-A was induced by phosphorylation and protein stabilization by HER-2/Neu rather than increased Aurora-A gene expression. Moreover, Aurora-A stabilization induced an increase of HER-2/Neu expression in breast cancer cells defining a novel positive feed-back loop between Aurora-A and HER-2/Neu oncogenic signaling pathways. Basal-like tumor cells over-expressing Aurora-A and HER-2/Neu underwent to loss of CD24 surface receptor, expressed epithelial to mesenchymal transition (EMT) markers and acquired stem cell-like properties such as mammospheres formation and self-renewal. Pharmacologic inhibition of HER-2/Neu signaling pathway with lapatinib in cancer cells over-expressing Aurora-A demonstrated that Aurora-A was downstream to HER-2/Neu in the development of EMT and stemness. To determine whether Aurora-A induced a cancer stem cell-like phenotype through development of chromosomal instability and tumor cell phenotypic heterogeneity, we performed a SKY analysis on the breast cancer cell lines employed in this study. Our results demonstrate that invasive breast cancer cells harboring a basal-like phenotype did not develop higher chromosomal instability compared to luminal non-invasive cancer cells, signifying that Aurora-A has a direct effect on the development of basal-like breast cancer cells with stemness properties. Importantly, pharmacologic inhibition of Aurora-A kinase activity with the small molecule MLN8237 restored CD24 expression and reversed EMT leading to the re-establishment of a luminal phenotype and suppression of distant metastases. Our findings implicate for the first time the mitotic kinase Aurora-A in the development and maintenance of CD24 negative basal-like tumor stem cells responsible for breast cancer recurrence and progression. These observations have also important implications for the development of molecular targeted therapies to inhibit Aurora-A kinase activity and stemness improving the overall survival of breast cancer patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 328. doi:1538-7445.AM2012-328