bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4572: Comparison of proteomic profiles in human plasma of smokers and nonsmokers using the iTRAQ approach
Authors: Bortner James D. , Richie John , Das Arunangshu , Liao Jason , Umstead Todd , Stanley Anne , Stanley Bruce , Belani Chandra , El-Bayoumy Karam
Published: February 2011

Lung cancer is a potentially preventable disease because it is strongly related to tobacco smoke. Although there have been great strides taken to inform the population of the severity of the risks associated with smoking, there are currently 43.4 million Americans that smoke and 1.3 billion smokers worldwide. Therefore, it is important to develop biomarkers that can identify individual smokers and ex-smokers who are at especially high risk for lung cancer. Relative changes that take place in the human plasma proteome as a result of smoking may be playing an important role in the etiology of lung cancer. Thus, in this study we used a proteomic approach utilizing isobaric Tag for Relative and Absolute Quantitation (iTRAQ) to compare the plasma protein expression levels between healthy smokers and non-smokers. With this multi-plex non-gel based technique, up to 8 samples can be simultaneously labeled with isobaric tagging reagents, 113-119 and 121, for quantitation and identification by mass spectrometry. Plasma samples from seven white male smokers and seven white male non-smokers were distributed into two 8-plex iTRAQ sets. The 113 label in each set was used to label a pool consisting of each sample in the study in order to compare between the two 8-plex sets. Plasma from each subject was depleted of the 14 most abundant plasma proteins using an immunoaffinity column to search for lower abundant proteins. Based on our results, we identified 18 significant (p<0.05) differentially expressed proteins between smokers and non-smokers. The proteins that were down-regulated in smokers include sex hormone-binding globulin (SHBG), inter-α (globulin) inhibitor H3 (HC3), and vitronectin. The proteins that were up-regulated in smokers include the complement component 8, α, β and γ polypeptides, gelsolin, and mannose-binding protein C (MBP-C). Among those identified, SHBG, HC3, and gelsolin protein have been previously identified in smokers and have been suggested to be candidate markers for lung cancer. The results of this study show that cigarette smoking can influence the relative expression profile of plasma proteins. However, the relationship of these proteins to the development of lung cancer needs to be determined so that they can be further developed as biomarkers of disease risk.

Support: NCI PO1 70972

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4572.
Abstract 4585: Verification of prostate cancer biomarker candidates by nanoparticle-capture MRM
Authors: Fredolini Claudia , Luchini Alessandra , Meani Francesco , Russo Paul , Tamburro Davide , Douglas Temple , Longo Caterina , Gambara Guido , Zhou Weidong , Ross Mark , Novelli Francesco , Collura Devis , Muto Giovanni , Espina Virginia , Ornstein David , Liotta Lance A. , Petricoin Emanuel F.
Published: February 2011

Prostate adenocarcinoma (PCa) is highly diffuse between older men, and biomarkers that allow discrimination of cancerous pathologies from benign diseases, identification of clinical aggressive cancer with high specificity and monitor cancer progression are required. Recently we applied a new workflow for the discovery of candidate biomarkers based on hydrogel nanoparticle capture of LMW serum borne analytes. Comparing serum from pre and post prostatectomy patients we identified 112 candidates biomarkers. Our novel nanoparticle technology provides a rapid and near 100% efficient means to capture and greatly amplify the resting concentration of LMW protein analytes. Since most of these markers do not have well performing specific antibodies commercially available, we utilized MRM to verify their differential expression as first identified by Orbitrap-MS/MS.

Hydrogel nanoparticle serum pre-processing was used for both Orbitrap-LTQ based discovery and MRM verification such that the same analyte fractionation workflow was used but different downstream mass spectrometry. MRM was performed on a Thermo Quantum triple quadrupole-MS using the nanoparticle trapped analyte archive as the input for analysis. The following biomarker candidates were selected for MRM verification: Apolipoprotein E, Diacylglycerol kinase alpha, complement 1 subcomponent q B chain precursor, complement component 1 q subcomponent gamma polypeptide, serpin peptidase inhibitor, vitronectin precursor, angiotensinogen preproprotein, H4 histone family member J, fibronectin 1 isoform 2, orosomucoid 1 precursor, alpha-2-HS-glycoprotein, annexin I.

Several prostate candidate biomarkers were verified by Nanoparticle capture-MRM, thus providing data supporting the use and continued improvement of this novel biomarker discovery system for the identification of protein biomarker panels for early disease detection and therapeutic monitoring.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4585.
Abstract 4589: Changes in proteomic profiles with age in male rats using the iTRAQ approach
Authors: Das Arunangshu , Richie John , Bortner James , Aliaga Cesar , Stanley Anne , Stanley Bruce A. , El-Bayoumy Karam
Published: February 2011

Aging is an important risk factor for many diseases including prostate cancer (PC). Enhanced risk for disease may, in part, be due to aging-related changes in the proteome. Thus, a better knowledge of the changes in protein levels occurring with age would help in the early detection of the disease, as well as in monitoring chemopreventive intervention. In the present study we used male F344 rats, a commonly used aging and PC model, and compared protein levels in the blood plasma of young (4 months), mature (12 months), old (18 months) and very old (24 months) rats by a proteomic approach known as isobaric Tag for Relative and Absolute Quantitation (iTRAQ). In this multi-plex non-gel based technique samples can be simultaneously labeled with isobaric tagging reagents for quantitation and identification by mass spectrometry. Plasma samples were pooled from three animals and 50 μg of protein from each group was labeled for a 4-plex iTRAQ analysis. A total of seventy nine proteins were identified, thirty one with >99% confidence. Among those, the levels of thirteen proteins were changed significantly (p<0.05) with age and five of which with >99% confidence including serotransferrin, hemopexin, fetuin-A, fetuin-B and α-1-acid glycoprotein. Both age-specific and growth-development related changes were observed throughout the life span with maximum changes (either increases or decreases) being observed at 18 months. The results of this study show that age indeed can influence the expression profile of plasma proteins. The relationship of these proteins to PC is currently being investigated by analyzing their levels in different lobes of the prostate gland. Supported in part by R01 CA 127729-01

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4589.
Abstract 4590: Characterization of an epithelial micro-pattern within ducts and lobules of the human breast: The topography of estrogen receptor expression
Authors: Villadsen Rene , Fridriksdottir Agla Jael , Kim Jiyoung , Rønnov-Jessen Lone , Petersen Ole W.
Published: February 2011

Estrogen receptors (ERs) function as relays between oscillating levels of circulating estrogen and micro-environmental cues for growth and differentiation during breast development, function and carcinogenesis, yet the cells that express ERs are poorly understood. Using an antibody library and multicolour imaging, we have found that ERs, which are expressed in an apparently unpredictable manner throughout ducts and lobules of the human breast, are in fact patterned by an underlying compartment of cells with a unique differentiation program and topography. Furthermore, we show that while ER-occupancy of this compartment is highly variable from zero to full between individual ducts and lobules, its average size is relatively constant. Moreover, vacancy is associated with a slightly higher proliferative activity as revealed by staining for Ki-67 and the ER-compartment cells are able to form acinus-like colonies inside a reconstituted basement membrane. In human breast cancers staining for the ER-compartment program revealed that fifty percent of a sample of apparently ER-negative tumors in reality was composed of ER-compartment cells. These results establish an anatomical foundation for the expression of ER in the human breast and a link between a broader sense, normal ER-compartment and a subset of ER-negative albeit luminal human breast carcinomas.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4590.
Abstract 4600: The role of COUP transcription factors in Tamoxifen resistant breast cancer cells
Authors: Maniya Omar Z. , Zwart Alan , Chen Li , Xuan Jianhua , Clarke Robert , Riggins Rebecca B.
Published: February 2011

Breast cancer affects 180,000 women in the United States every year, killing over 40,000 of them. The most common type of breast cancer, which expresses the Estrogen Receptor (ER+), has been clinically treated with the antiestrogen Tamoxifen (TAM) for decades. However, up to 35% of patients taking TAM may develop resistance to the drug. This may be related to the fact that ER+ breast cancers are divided into Invasive Lobular Carcinomas (ILC) and Invasive Ductal Carcinomas (IDC), which may have different mechanisms for TAM resistance. Thus, TAM resistance represents a formidable challenge in the treatment of breast cancer.

TAM is an antagonist of estrogen receptor alpha (ERalpha), and prevents estrogen-dependent growth signaling in breast tissues. We have published that overexpression of the orphan nuclear receptor, estrogen related receptor gamma (ERRgamma), leads to TAM resistance in ER+ breast cancer cell lines derived from ILC. We have recently found that ERRgamma is also overexpressed in the IDC-derived RR TAM resistant cell line (but not in other models of TAM resistance). This leads us to hypothesize that other orphan nuclear receptors may have been overlooked in prior studies of TAM resistance, and that different members of the nuclear receptor superfamily play unique roles in ILC- and IDC-derived TAM resistant breast cancer.

To test this hypothesis, we profiled mRNA expression of the nuclear receptor superfamily in two pairs of TAM sensitive and resistant cells: the IDC-derived MCF7 and RR, and the ILC-derived MDA-MB-134VI and TAM-R. Expression of two members of the COUP transcription factor family was significantly changed. NR2F1 (COUP-TFI) was highly overexpressed in RR cells, and NR2F2 (COUP-TFII) was highly overexpressed in both RR and TAM-R cells relative to sensitive controls. These findings are highly significant as COUP transcription factors have been implicated in embryonic stem cell pluripotency, and NR2F1 has been previously shown to interact with and enhance ERalpha activity through increasing its phosphorylation by MAPK. Therefore, overexpression of NR2F1 (and possibly NR2F2) may counteract the antagonistic effects of TAM and represent a novel pathway for TAM resistance in lobular and ductal ER+ breast cancers. Ongoing studies include gel electrophoresis to determine whether this overexpression is observed at the protein level and siRNA knockdown experiments to determine the impact of NR2F1 and NR2F2 on ERalpha phosphorylation and activity, TAM resistance, cell death, and overall survival.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4600.
Abstract 4601: XBP1 regulated function of c-MYC and BCL2 in antiestrogen resistance in breast cancer
Authors: McDaniel Lauren M. , Shajahan Ayesha N. , Clarke Robert
Published: February 2011

Breast cancer is one of the most prevalent types of cancer in the United States. In fact, one in eight women will be diagnosed with breast cancer at some point in their lifetime. Approximately, 70% of newly diagnosed cases of invasive breast cancer are estrogen receptor-α positive (ER+) and in these patients, the disease is often managed by antiestrogen therapy. A barrier in successfully treating some of these patients is their tumors’ ability to develop resistance to the antiestrogens. Previously, we have shown that one mechanism though which breast cancer cells become resistant to antiestrogens is by up-regulating the X-Binding protein 1 (XBP1). Specifically, the presence of the spliced form of XBP1, XBP1(S), confers resistance to breast cancer cells by inhibiting apoptosis. Overexpression of XBP1(S) in MCF-7 breast cancer cells also up-regulates the expression of c-MYC, a transcription factor that is involved in cell proliferation and cell survival, and BCL2, an anti-apoptotic molecule. c-MYC can confer partial resistance to the antiestrogen ICI 182, 780, and can cooperate with and/or be regulated by BCL2. We are investigating the role of XBP1 in regulating c-MYC and BCL2 in antiestrogen resistance. We hypothesize that co-inhibition of c-MYC and BCL2 will restore antiestrogen sensitivity to resistant breast cancer cells. We are using two MCF-7 variants: LCC9 cells that are an estrogen/antiestrogen resistant (ICI 182, 780 resistant and tamoxifen cross-resistant) and LCC1 cells that are estrogen/antiestrogen sensitive. Using small molecule inhibitors or lentiviral short hairpin RNA particles or we are specifically inhibiting our target molecules (c-MYC and BCL2) and testing the effects on antiestrogen responsiveness. Our preliminary data indicate that under basal condition, the antiestrogen resistant LCC9 cells are more sensitive to 10058-F4 (c-MYC inhibitor) and ABT-737 (BCL2 inhibitor) compared to LCC1 cells. Thus, in LCC9 cells, c-MYC and BCL2 clearly play important roles in cell survival. Our results will elucidate how XBP1 signals through c-MYC and BCL2 to confer endocrine resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4601.
Abstract 4602: Effect of estrogen receptor antagonistic drugs on expression and regulation of fatty acid synthase (FAS) in ER+ breast cancer cell lines
Authors: Atefi Mohammad S. , Zheng Cristina , Atefi Zahra , Slamon Dennis J.
Published: February 2011

Expression of estrogen receptor (ER) is detected in more than sixty percent of breast tumors. High levels of Fatty acid synthase (FAS) have been detected in aggressive breast cancers. Due to its high level of expression, FAS has been considered as a tumor marker and a therapeutic target. In normal rat liver, increase in expression of FAS requires glucose and a combination of hormones. Activity of FAS is indirectly regulated by the upstream signaling factor AMPK. AMPK is considered to be the main regulator of cellular metabolism. Upon activation, AMPK down regulates FAS activity and other ATP consuming pathways. In this study, we investigated the possible regulatory role of estrogen receptor (ER) signaling on FAS level and activity. In a panel of ER+ cell lines, very high levels of FAS expression was observed in the majority of the cell lines. The highest levels of FAS were in the ER+/HER2+ cell lines, HCC1419, BT474 and MDA-MB-361, respectively. However, in two other ER+/HER2+ cell lines, UACC732 and UACC812, the expression was similar to or even lower than the HER2 negative cell lines. Three HER2 negative cell lines in the panel also showed very high levels of FAS expression. Upon incubation of the cells in the absence of serum and phenol red condition, a general decrease in the FAS levels was observed that was more evident in HER2 negative cell lines. In the same condition, the effects of ER inhibitory drugs, fulvestrant and tamoxifen, on FAS and p-AMPKα levels were investigated. In ER+ cell lines that contain high levels of p-AKT, ER inhibitors did not affect levels of FAS and p-AMPKα. However, in some cell lines with lower levels of active AKT, tamoxifen and fulvestrant induced changes in FAS and p-AMPKα levels that were variable from cell line to cell line. In MCF-7 cell line, which is highly sensitive to ER inhibitors, both tamoxifen and fulvestrant induced lower FAS and higher p-AMPKα levels. Contrary to this observation, tamoxifen induced higher FAS and lower p-AMPKα levels in T-47D and KPL-1 cell lines. Fulvestrant induced a similar effect in ZR-75-1 cell line. This inducing effect of ER antagonists on FAS perhaps reflects one of the mechanisms that cause the elevation of blood lipids in post menopausal and tamoxifen treated women.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4602.
Abstract 4604: Roscovitine modulates ERα/ERβ ratio: A novel means to target hormonal therapy resistance
Authors: Nair Binoj Chandrasekharan , Morales Mary , Nair Hareesh Babu Bhaskaran , Vallabhaneni Sreeram , Li Rong , Tekmal Rajeshwar Rao , Vadlamudi Ratna K.
Published: February 2011

Current endocrine therapy for ER+ve breast cancer involves modulating ERα-pathway using either ERα-antagonists or aromatase inhibitors. Despite the positive effects, de novo and/or acquired resistance to endocrine therapies frequently occur. Although mechanisms for hormonal therapy resistance remains elusive, emerging data implicate ER-growth factor signaling cross talk, and alteration in ER subtypes as the major causes of resistance. Most downstream events in the resistance pathways converge upon modulation of cell cycle regulatory proteins; the most conspicuous of which is the activation of Cyclin Dependent Kinase 2 (CDK2) pathway. Roscovitine is one of the most frequently studied and used CDK2 inhibitor. In this study, we examined whether roscovitine confers tumor suppressive advantage to therapy resistant breast epithelial cells by inhibiting CDK functions using three therapy resistant model cells; (a) MCF-7-Tam (acquired Tamoxifen resistance model); (b) MCF-7-CA-LTLT (acquired Letrozole resistance model); (c) MCF-7-HER2 (ER-growth factor signaling cross talk model). Roscovitine at 10 μM concentration substantially reduced the growth of all three resistant model cells in cell proliferation and foci formation assays. FACS analysis revealed that roscovitine treatment increased proportion of cells in G2-M. To increase the drug efficacy, we have developed PLGA nanoparticles containing roscovitine and this mode of delivery significantly reduced the dosage needed to see the growth inhibitory effect in therapy resistant cells to 500 nM. To determine the mechanism, we have determined the relationship between CDK activity, expression of ERα, β subtypes, ER-coregulators, and expression of ER target genes in the presence or absence of roscovitine in resistant cells. Roscovitine substantially affected CDK2 activity and decreased cyclin A levels in the model cells. These studies also revealed an unexpected discovery that CDK inhibitor roscovitine has potential to alter the ratio of ER isoforms with preferential upregulation of ERβ expression with concomitant down regulation of ERα, its coregulators including AIB1 and PELP1. Since many advanced and therapy resistant tumors exhibit loss or reduced expression of ERβ, dual action of roscovitine will provide a novel drug to inhibit both CDKs and to increase the expression of tumor suppressor ERβ. Since deregulation of cell cycle machinery and estrogen receptor signaling contributes to resistance, roscovitine a drug that attacks both these pathways will serve as a double-edged sword to interfere with the resistance mechanisms.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4604.
Abstract 4605: Combinatory use of synthetic and natural ER beta modulators in treating hormonal-therapy-resistant breast tumors
Authors: Ganapathy Manonmani , Nair Hareesh Babu Bhaskaran , Kirma Nameer , Krishnegowda Naveen , Vadlamudi Ratna , Mary Tagliaferri , Isaac Cohen , Tekmal Rajeshwar
Published: February 2011

Although systemic hormone therapies that either block local estrogen production by aromatase inhibitors (AI) such as letrozole or block actions of estrogen/ER actions by anti-estrogens like tamoxifen are well tolerated, the development of resistance to these agents is still a major concern. We have investigated whether treatment of letrozole resistant breast cancer cells with selective ER beta agonists (synthetic and of natural origin) in combination with letrozole restored sensitivity to this AI. We performed similar combination treatment of tamoxifen resistant cells with ER beta agonists and tamoxifen. To test whether combination therapy restores sensitivity to letrozole, we have treated letrozole resistant breast cancer cells LTLTca, derived from MCF-7-aromatase tumors resistant to letrozole] with either letrozole alone or with a combination of letrozole and DPN. Compared to single agents, combination treatment not only restored sensitivity to letrozole but also resulted in decreased cell proliferation and increased apoptosis as well as increased ERβ and decreased ERα levels in these cells. When we tested the growth of letrozole resistant breast tumors using a postmenopausal xenograft model, combination therapy not only restored sensitivity to letrozole but resulted in decreased tumor growth. Long term treatment using nanoparticle- encapsulated DPN and letrozole resulted in a significant and sustained decrease of tumor growth. On the other hand, nanoparticle-encapsulated letrozole treatment resulted in tumor resistance to therapy in13 weeks. In search for better and safer therapeutic modulators for ER, We have tested Liquiritigenein and Nyasol as selective natural ER beta agonists. Effects of these compounds on cell growth was tested in different breast cancer cells including MCF-7, MDA-MB-231, tamoxifen resistant MCF-7, and LTLT-Ca. In tamoxifen resistant MCF-7 breast cancer cells 15uM of liquiritigenin and Nyasol showed ∼70% and 90% of cell death, respectively. Different biological activities of liquiritigenin have been reported including anti-inflammatory activity. Combination of liquiritigenin and Nyasol with letrozole diminished cell growth in MCF-7, MCF-TAM, MCF-Aro and LTLT-Ca cells. Combination therapy using ER beta agonists like DPN, liquiritigenin and nyasol may offer superior response and restore the sensitivity to the current AI and anti-estrogens. Our study suggest a novel role of ER beta agonists as valuable therapeutic modulators to treat endocrine sensitive as well as resistant tumors. (Supported by NIH/NCI CA 750118 & P30 CA 54174, RRT)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4605.
Abstract 4614: Progesterone receptor and ErbB-2 crosstalk in breast cancer
Authors: Béguelin Wendy , Flaqué María C. Díaz , Cayrol Florencia , Proietti Cecilia J. , Rivas Martín A. , Tkach Mercedes , Charreau Eduardo H. , Schillaci Roxana , Elizalde Patricia V.
Published: February 2011

Progesterone receptor (PR) and ErbB-2, a member of the ErbBs family of receptor tyrosine kinases, play key roles in breast cancer development. Here, we identified a new mechanism of PR and ErbB-2 interaction in breast cancer cells. Our findings demonstrated that progestin stimulates the rapid activation of ErbB-2, its nuclear translocation, and a striking nuclear colocalization of ErbB-2 with signal transducer and activator of transcription 3 (Stat3) in human T47D breast cancer cells and C4HD cells, from a murine progestin-dependent mammary tumor. By using quantitative chromatin immunoprecipitation (ChIP) and sequential ChIP, we demonstrated that progestin induces the recruitment of Stat3 and ErbB-2 to Stat3 response elements (GAS sites) in the cyclin D1 promoter, which lacks ErbB-2 response elements (HAS sites). Through a series of experimental approaches including transient transfection with cyclin D1 promoter luciferase constructs and RNA interfering-reconstitution strategies employing functional ErbB-2 and Stat3 mutants, we revealed ErbB-2 role as a transcriptional coactivator of Stat3 in progestin-induced cyclin D1 promoter activation. Inhibition of ErbB-2 nuclear migration by transfection of breast cancer cells with the ErbB-2 nuclear localization domain mutant ErbB-2ΔNLS (Mol Cell Biol, 24: 11005-11018, 2005), which we here found that functions as a dominant negative inhibitor of endogenous ErbB-2 nuclear translocation, resulted in complete abrogation of progestin-induced in vitro and in vivo breast cancer growth. Our findings reveal a novel therapeutic intervention in PR- and ErbB-2- positive breast tumors via the specific blockage of ErbB-2 nuclear translocation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4614.
Abstract 4618: Effect of restoration of estrogen receptor-α (ER α) expression on drug sensitivities of gastric cancer cell lines
Authors: Woo In Sook , Kim Kyung Ae , Lee Kyu Sang , Byun Jae Ho , Han Chi Wha
Published: February 2011

Introduction: The significance of expression of estrogen receptor-α (ER α) and hormone manipulation is not established in gastric cancer. Positivity of ER α expression in gastric cancer ranges from 0-67%. We previously reported that promoter methylation of ER α is associated with loss of ER α in gastric cancer cell lines. The aim of this study was to examine the changes in sensitivity to tamoxifen and 5-fluorouracil (5-FU) after induction of ER α by the DNA methyltransferase-1(DNMT1) inhibitor 5′-aza-deoxycytidine (AZA) and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA), following gene transfection of ER α into gastric cancer cell lines. Methods: ER α-negative gastric cancer cell lines including SNU-484, −638, and −71 were treated with AZA (10 μmol/L) and TSA (500 nmol/L). An ER α-encoding plasmid was transfected into an ER α-negative breast cancer cell line, MDA-MB-231, and gastric cancer cell lines. Cell proliferation was assayed in ER α-negative and -restored cell lines treated with tamoxifen (0, 1, 2.5, 5, 10, 15, 20, and 25 μM) or 5-FU (0, 0.02, 0.2, 2, 20, 200, and 1,000 μM). Results: Restoration of ER α expression in ER α-negative gastric cancer cell lines after treatment with AZA and TSA was confirmed by RT-PCR. Growth inhibition was more prominent in gastric cancer cell lines transfected with the ER α gene (with tamoxifen at a concentration of more than 15 μM) or treated with trichostatin (with tamoxifen at a concentration of more than 20 μM) than in ER α-negative gastric cancer cell lines. No differences in sensitivity to 5-FU were observed regardless of whether ER α was present or absent. Conclusion: The DNMT inhibitor AZA and the HDAC inhibitor TSA, together with tamoxifen, deserve further investigation as new targeted therapies in ER α-negative gastric cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4618.
Abstract 4621: Downregulation of Protein Kinase D1 in hepatocellular carcinoma (HCC): Potential as a diagnostic/prognostic molecular marker
Authors: Prakash Preethi , Freiz Matt , Bohlmeyer Teresa , Koch Micheal R.D , Chauhan Subhash C. , Jaggi Meena
Published: February 2011

OBJECTIVE: Protein Kinase D1 (PKD1) plays an important role in several cellular processes such as cell proliferation, oxidative stress signaling, adhesion and motility. PKD1 is of particular interest in diagnosis, prognosis and treatment of human diseases because of its enzymatic activity and susceptibility to successful therapeutic targeting. In our earlier study we have demonstrated suppression of PKD1 expression in advance stage prostrate cancer. This led us to analyze PKD1 expression patterns in various other cancer tissue samples.

MATERIAL AND METHODS: Tissue microarrays containing various cancer samples with corresponding normal tissues were used to investigate the expression profile of PKD1 using immunohistochemistry (Biocare Medical, Concord, CA). Further studies on PKD1 expression profile in hepatocellular carcinoma (HCC) were carried out using tissue microarray (TMA) containing HCC samples (n=70) with normal tissues (n=4) and a HCC TMA with hepatitis B viral history (HBV) containing HCC samples (n=100) with normal tissues (n=4) (AccuMax Array). Immunostaining was graded by two independent pathologists. Intensity and extent of staining scores were multiplied to obtain the composite score (CS) of each sample.

RESULT: Differential expression pattern of PKD1 was observed in brain, skin, lung, kidney, thyroid and ovarian cancer samples. Along with stomach and colon cancer, HCC samples showed a marked downregulation of PKD1 expression compared to their respective normal tissues. PKD1 expression profile was further evaluated in a large cohort of HCC samples. Overall, HCC samples showed a significant (p>0.05) downregulation (Mean CS=6.0) of PKD1 expression compared to normal liver samples (Mean CS=11.4). Among cancer samples, advanced stage samples showed a relatively lower PKD1 expression (Mean CS=5.4) compared to early stage (Mean CS=6.6) HCC samples. Similarly, advance stage HCC samples with HBV showed a lower PKD1 expression (Mean CS=6.9) compared to early stage samples (Mean CS=9.6) and non neoplastic samples (Mean CS=12).

CONCLUSION: The results of this study suggest suppression of PKD1 expression in HCC. In view of the limitations of the available diagnostic/prognostic studies for detection of HCC, the aberrant expression of PKD1 may serve as a novel biomarker for the diagnosis/prognosis of hepatocellular carcinoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4621.
Abstract 4622: PAM4-antigen levels in the serum of patients with pancreatic carcinoma: Early detection of disease and correlation with responses to radioimmunotherapy
Authors: Gold David V. , Newsome Guy , Modrak David E. , Horne Heather , Wegener William A. , Goldenberg David M.
Published: February 2011

Background: A serum-based enzyme immunoassay (EIA) employing MAb-PAM4 demonstrates high specificity and sensitivity for detection of advanced pancreatic carcinoma (PC). We are currently exploring use of the immunoassay as a means to detect early-stage PC, as well as detection of nascent disease progression or relapse during routine post-treatment follow-up. We now also report initial results that provide correlation of serum PAM4-antigen levels with responses to radiolabeled PAM4 therapy.

Methods: The PAM4-based EIA was employed for detection and quantitation of antigen in the serum of pancreatic carcinoma patients with known stage of disease (N=68). An additional 11 patients with stage-4 disease who participated in an ongoing phase-1b clinical trial to evaluate the combination of 90Y-hPAM4 IgG, (clivatuzumab tetrexate) radioimmunotherapy with low-dose, radiosensitizing, gemcitabine (Gem) were also examined. Serum specimens from this latter group were collected at baseline prior to treatment, at the end of the 4-week treatment cycle, and again at 4 weeks post-treatment (week 8 from baseline), when a CT scan was performed to determine tumor response.

Results: Overall, the sensitivity of the immunoassay for detection of PC was 81%, with a 5% false-positive rate for the healthy volunteers (N=19). When examined by stage of disease, sensitivity rates were 91%, 86% and 62% for stages 3/4 advanced disease, stage-2, and stage-1 PC, respectively. For those patients who had treatment with 90Y-hPAM4 + Gem, a decrease in antigen levels greater than 40%, observed at the end of the treatment cycle (4 weeks), with sustained decline in the antigen levels at the 8-week post-treatment evaluation, provided presumptive evidence of tumor response, either stable disease (SD, N=2) or partial response (PR, N=5) by CT RECIST criteria. None of the patients with progressive disease (N=4) had a sustainable decrease in serum levels of PAM4-antigen, whereas all of the responders did. The median decrease in serum PAM4-mucin levels at the 8-week follow-up evaluation timepoint was 54% (range: 47% - 98% decrease), with a median tumor response (only SD and PR responders) being a decrease in tumor size of 37% (range: 46% decrease to 4% increase), from a baseline median primary tumor size of 10.0 cm (range 2.6 - 12.1 cm).

Conclusions: These results suggest that the PAM4-based immunoassay should be further evaluated for use in the detection of early pancreatic carcinoma, as well as to evaluate tumor response to therapy. (Supported in part by grant CA096924 from the NIH.)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4622.
Abstract 4624: Differentiation of female donors classified as “normal”, with benign disease and with breast cancer based on blood RNA signature
Authors: Somiari Richard I. , Russell Stephen , Bharathan Seema , Lyons-Weiler James , Somiari Stella A.
Published: February 2011

Breast cancer still imposes significant healthcare burden on women worldwide. Mammography is currently the benchmark for screening, but mammography may not; a) detect all early stage breast cancers especially in young women with dense breast, b) unequivocally distinguish between benign disease and cancer and c) be beneficial to people in remote and underserved areas without mammograms. Biomarkers could complement mammography because of the potential to enable earlier detection, accurate differentiation between non-cancerous and cancerous breast lesions, and allow screening of a specimen like blood collected from a remote site. As a step towards developing a biomarker-based blood test for early detection of breast disease, we have used the branched-DNA multiplex technology to measure the levels of 24 candidate biomarkers in blood of fully informed and consented female donors classified into three broad categories; normal, with breast cancer (BC), and with benign breast disease (BD). To determine if the groups can be identified using the levels of the 24 candidate biomarkers we trained prediction models based on the data without background subtraction and applied the same training process on background subtracted data. Next we used an in-house developed software, to identify the optimal transformation and normalization methods. Due to the small sample size available for the current study, we generated 1,000 cases and 1,000 controls by simulating the original data via a distributional sampling model, and used the simulated data as the test validation set (TVS). Three prediction models were then applied to the original data and simulated TVS, namely: in-house GA optimized k of m, Random Forest (Salford System), Logistic Regression (SAS). Random Forest proved to be the best model for group prediction displaying sensitivities and specificities above 90%. This presentation will discuss the extent to which the levels of the biomarkers in blood allowed the identification of donors classified as “normal” and with “breast disease”.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4624.
Abstract 4635: FK506 binding protein 51 is a possible novel tumoral marker
Authors: Romano Maria Fiammetta , Romano Simona , D'Angelillo Anna , Luna Elvira De , Staibano Stefania , Mascolo Massimo , Bisogni Rita
Published: February 2011

Very recently, we have identified FK506 binding protein 51 (FKBP51), an immunophilin phisiologically expressed in lymphocytes, as a protein associated with malignant melanoma. Its expression correlates with tumor aggressiveness and is maximal in metastatic lesions. We have also demonstrated that FKBP51 is a factor of resistance to genotoxic agents, including anthracyclins and ionizing radiations, because it promotes NF-kB activation. Aim of the present study was to investigate FKBP51 expression in solid tumors. Overall 60 samples, stored in our Patology section, of the following tumors: breast, colon, lung, pancreas, ovary and prostate (10 for each tumor), and a comparable number of normal tissue samples, were analysed for FKBP51 expression by immunohistochemistry. An intense signal was observed in all prostatic and ovarian adenocarcinomas analyzed, but only in a proportion of pancreatic cancer samples analyzed (mostly, esocrine pancreas tumors). Furthermore, an intense nuclear signal was observed in colon and lung adenocarcinomas. Immunohistochemistry was negative in breast cancer. Normal tissues of the same histotype showed very low or undetectable levels of FKBP51. We also measured FKBP51 mRNA levels in deparaffinized tissues from breast, pancreas, prostate and lung samples, both normal and tumoral, by Realtime PCR. All normal tissues analyzed, except for prostate, displayed mRNA levels lower than those of peripheral blood mononuclear cells (PBL). FKBP51 levels in prostate and PBL were similar. Increases in mRNA levels, from 3 to 30 folds, were found in prostate, esocrine pancreas and lung cancers, compared to normal tissue of the same histotype. Very low FKBP51 mRNA levels were found in breast cancer. In conclusion, our preliminary results show that FKBP51 expression increases in different solid tumors, including ovary, prostate, lung, colon, and esocrine pancreas, in comparison with the normal tissue counterpart. This study support the conclusion that deregulated FKBP51 can play a role in malignant transformation.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4635.
Abstract 4638: Over-expression of p19 protein in human gastric carcinoma and its clinicopathological significance
Authors: Tsai Ming-Ming , Wang Chia-Siu , Lin Kwang-Huei
Published: February 2011

Gastric cancer is the fifth leading cause of cancer-related death in Taiwan. Identification of gastric cancer related factor is essential to increase patient survival. P19 protein was identified using two-dimensional electrophoresis (2-DE) combined with Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometer (MALDI-TOF MS) and isobaric tag for relative and absolute quantitation (iTRAQ). We evaluated the p19 protein expression as a potential biomarker of gastric cancer since it aberrantly expressed in gastric cancer and associated with metastasis. Tumor tissue and matching noncancerous mucosa were obtained from 60 patients immediately after gastric resection. P19 protein expression levels were determined by quantitative reverse transcriptase-polymerase chain reaction (Q-RT-PCR) and Western blot. The expression of p19 was up-regulated 76.3% of 55 patients, or 45% of 60 patients with gastric cancer in tumor tissues (compared with adjacent non-tumor tissues) at mRNA or protein levels, respectively. Paraffin-fixed tumor tissues were used for immunohistochemistry (IHC) study in 110 patients with gastric cancer. The p19 protein was up-regulated in about 58% tumor tissues, however, not in plasma. The clinic-pathological correlation of p19 protein was also valuated. The higher p19 expressions were significantly associated with lymph node metastasis, and were significantly higher in the stages of III and IV than those in the stages I and II. The depth of wall invasion was significantly greater in the stage of (T3 and T4) compared to the (T1 and T2). Five-year survival of patients with lower p19 expression was significantly better than those patients with a higher expression. To better understand the function of p19 protein in human gastric cancer cells, the isogenic p19 protein overexpressing cell lines (AZ521, SC-M1) were prepared. The proliferative, migratory and invasive abilities were increased in the p19 overexpressing cell lines. The results indicate the p19 protein is a potential prognostic marker for gastric cancer and its function associates with cell invasion.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4638.
Abstract 464: Hypoxia-regulated Delta-like 1 homologue (DLK1) enhances cancer cell stemness and tumorigenicity
Authors: Kim Yuri , Lin Qun , Zelterman Daniel , Yun Zhong
Published: February 2011

Tumor hypoxia, i.e. O2 deficiency, is a common feature of solid tumors and predicts poor clinical outcomes. Tumor hypoxia is associated with increased aggressiveness and resistance to cancer therapies. Hypoxic tumors appear to be poorly differentiated and express stem/progenitor cell genes. It remains unclear, however, whether and how hypoxia regulates cancer cell differentiation and maintains cancer cell stemness. In this study, we demonstrate that hypoxia increases the expression of delta-like 1 homolog (DLK1), a transmembrane protein mainly expressed in stem/progenitor cells. Both HIF-1α and HIF-2α can bind to the DLK1 promoter. Using neuroblastoma (NB) cells as a model, we have found that DLK1 is highly expressed in undifferentiated, but not in differentiated, NB tumor cells. Downregulation of DLK1 expression by siRNA interference results in spontaneous differentiation, whereas overexpression of DLK1 inhibits retinoic acid-induced differentiation. We have further found that DLK1 is required for maintaining tumor sphere growth, a key feature reflecting the cancer stem cell-like characteristics. Importantly, we have demonstrated that loss of DLK1 expression leads to significantly reduced clonogenic growth in vitro, suppressed tumor take and growth of tumor xenografts in vivo. Furthermore, we have found that the intracellular domain of DLK1, especially its two conserved putative phosphorylation sites (Tyrosine 339 and Serine 355), is required for its biological functions. In summary, our results clearly demonstrate that DLK1 has the ability to regulate all the key aspects of cancer stem cell-like characteristics, i.e. tumor cell differentiation, self-renewal (tumor sphere growth), clonogenicity, and tumorigenicity. Because elevated DLK1 expression is also found in many tumor types, our observations suggest that hypoxia and DLK1 may constitute an important stem cell pathway for the regulation of cancer stem cell-like functionality and tumorigenicity by the tumor microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 464.
Abstract 4642: Risk stratification of bladder cancer patients using a panel of 5 prognostic protein markers
Authors: Fristrup Niels , Ulhøi Benedicte P. , Borre Michael , Hartmann Arndt , Ørntoft Torben F. , Dyrskjøt Lars
Published: February 2011

The majority of patients with bladder cancer are initially diagnosed with noninvasive or superficially invasive urothelial tumors. These tumors form a heterogeneous group, spanning from benign low grade Ta tumors that rarely progress to high grade T1 tumors with concomitant CIS that progress in up to 60% of cases. No biomarkers have yet been accepted in clinical routine for predicting the individual disease courses of these patients. Previously, we have identified gene expression signatures for prediction disease outcome for patients with non-muscle invasive bladder cancer. The aim of the present study was to validate key candidate markers from these signatures on the protein level. Five genes were selected for investigation: CTSE, BIRC5, TRIM29, PLK1 and SERPINB5.

A total of 289 primary non-muscle invasive urothelial tumors (182 pTa, 101 pT1 and 6 CIS) were chosen for tissue microarray construction. 118 patients showed later disease progression to pT2-4 bladder cancer with a median time to progression of 22 months. None of the remaining 171 patients showed later disease progression within the follow-up period with a median follow-up of 93 months. Protein expression was investigated using immunohistochemistry.

We found that high expression of BIRC5 and PLK1 identified patients with an increased risk of progression; (HR=2.47 (1.57-3.87), P<0.001 and HR=1.52 (1.00-2.29), P=0.048 respectively). Whereas high expression of CTSE, SERPINB5 and TRIM29 identified patients with a reduced risk of progression; (HR=0.60 (0.39-0.93), P=0.02), (HR=0.58 (0.38-0.89), P=0.01) and (HR=0.38 (0.20-0.73), P=0.003 respectively).

We found all five proteins to be independent prognostic variables for predicting disease progression to muscle invasive bladder cancer when stratifying for other known clinical and histopathological risk factors. Consequently, we successfully transferred key markers from the gene expression signatures to the protein level. This marker panel is currently being validated in a new series of pT1 bladder tumors and the results from this independent validation set will also be presented at the meeting.

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

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4642.