Cap Tissue Research Articles

Abstract A64: ASPORIN expression is associated with prostate cancer progression and bone metastasis

Abstract Being one of the most common male cancers in western countries, prostate cancer (CaP) is also considered as a leading cause of cancer-related death. Advanced CaP patients display characteristically bone metastasis, a devastating and incurable phase which leads to patient morbidity and mortality. It is widely recognized that bone metastases are intimately associated with bone remodeling, resulting from interactions between tumor cells, tumor-associated stromal cells and bone cells. The understanding of molecular events involving in tumor-stroma, tumor-bone cells cross-talks may help to identify new mechanism in prostate cancer progression, new diagnostic markers as well as therapeutic targets. Here, we report for the first time the expression profile of ASPORIN (ASPN) in human primary prostate cancer and associated bone metastasis. ASPN is a member of the class I small leucine-rich proteoglycan (SLRP) family. This matrix protein has been showed to be involved in the inhibition of Transforming Growth Factor Beta (TGFβ) signaling pathway in cartilage, and its capacity to bind type-I collagen and calcium suggests its role in bone-forming osteoblast collagen mineralization processes. By RT-PCR, our results showed that ASPN gene transcript was up-regulated in human primary CaP tissues. Immunohistochemical staining of ASPN protein reveals its expression in CaP tumor-associated stromal cells. Using an experimental mouse bone metastasis model, we showed strong ASPN expression not only in tumor-associated stromal cells, but also in CaP tumor adjacent mouse osteoclasts and osteoblasts. Similar ASPN expression profile was identified in human CaP bone metastasis samples. Further studies of immunohistochemical staining on CaP tissue microarray suggested ASPN stromal expression profile was significantly associated with disease progression, which was confirmed by the results of metaanalysis of 167 nonmalignant, primary and metastatic prostate cancer DNA microarrays. Taken together, all these findings strongly suggest that ASPN could be potentially used to predict the aggressiveness of prostate cancer and bone metastasis. Meanwhile, this secreted protein localized in tumor cell microenvironment may influence prostate cancer cell behavior. Since ASPN has been shown to be involved in TGFβ signaling regulation in cartilage, it could also be involved in the osteoblastic/mixture lesion progression in bone in which this pathway plays an important role. Citation Format: Tian V. Tian, Sebastien Flajollet, Martine Duterque-Coquillaud, Diane Goltz, Sven Perner, Anne Flourens, Nathalie Tomavo, Edith Bonnelye, Yvan de Launoit, Nicolas Wernert, Xavier Leroy. ASPORIN expression is associated with prostate cancer progression and bone metastasis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A64.

Abstract C43: Prognostic value of hypoxia-associated genes in prostate cancer

Abstract Tumor-associated hypoxia has been associated with malignant progression, metastasis and resistance to therapy. We studied the role of oxygen tension (pO2) in modulating the properties of cultured human prostate cancer (CaP) cells. In air, where oxygen tension (pO2) is approximately 20 kPa, cultured CaP cells expressed lower levels of transcripts associated with cancer and urologic disease; reducing pO2 to 2 kPa made these levels comparable to those in surgical CaP specimens suggesting that hypoxically cultured cells, rather than normoxically cultured cells, express genes at levels akin to those in tumor cells in situ. In human primary CaP tissue, transcripts of hypoxia-associated genes DLG7, CCNB1 and HMMR were overexpressed and associated with Gleason score and disease prognosis; this implies the potential prognostic value of these transcripts. While hypoxia affected the CaP cell proteome mostly quantitatively, it did affect expression of tumor–associated antigens and suggested the potential therapeutic relevance of the hypoxic antigenic landscape. Overall, our results suggest that hypoxia modifies cellular properties of cultured CaP cells towards a phenotype more similar to tumor cells in situ. Introduction of pO2 as a variable can constitute a tool for the identification of more effective prognostic biomarkers and development of better therapy for CaP and, possibly, other tumors. Citation Format: Christian R. Gomez, Stanimir Vuk-Pavlovic, Farhad Kosari, Jan Marie Munz, Claire Schreiber, Gaylord Knutson, Cristine Charlesworth, R. Jeffrey Karnes, John Cheville, George Vasmatzis. Prognostic value of hypoxia-associated genes in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C43.

A new method to apply and quantify bruising sensitivity of button mushrooms

Mushrooms are prone to develop brown discolouration due to bruising caused by mechanical damage during harvest, which leads to reduced quality of the mushrooms. In order to study the mechanism behind discolouration and to breed for bruise-related browning resistant strains, a high throughput bruise application method and reliable quantification of browning sensitivity is essential. In this study a new bruising device was developed to bruise mushrooms in a fast and reproducible way. The bruising device can apply damage to the cap tissue of button mushrooms by a slip-shear sliding process. The severity of the bruise was quantified with a computer imaging system combined with a newly developed software programme. A protocol was developed to obtain the most reliable and reproducible method to compare browning sensitivity of different Agaricus bisporus strains. For this purpose, the extent of damage applied, time between harvest and bruising, developmental stage of the mushrooms and flushes were taken into account. The new method of bruising and image based quantification was subsequently applied to a collection of wild, commercial and hybrid A. bisporus strains and a distinction could be made between bruise-related browning sensitive mushrooms and tolerant mushrooms.

The prostate cancer-up-regulated long noncoding RNA PlncRNA-1 modulates apoptosis and proliferation through reciprocal regulation of androgen receptor

ObjectiveEmerging evidences implicate long noncoding RNAs (lncRNAs) are deregulated in cancer development. The purpose of the current study is to investigate the role of new lncRNA, named PlncRNA-1, in prostate cancer (CaP) pathogenesis. Materials and methodsIn this study, real-time q-PCR was used to demonstrate the expression of PlncRNA-1 in 16 pairs CaP tissues and matched normal tissues, 14 pairs CaP tissues and BPH tissues, 4 CaP cell lines, including LNCaP, LNCaP-AI, PC3, and C4-2, and 2 normal prostate epithelial cell lines RWPE-1 and PWR-1E. After PlncRNA-1 was suppressed by siRNA in LNCaP and LNCaP-AI cell lines, cell proliferation and apoptosis were assessed using CCK-8 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). After PlncRNA-1 and AR was suppressed by siRNA in LNCaP and LNCaP-AI cell lines, real-time q-PCR and Western blotting were used to measure reciprocal regulation of PlncRNA-1 and AR. ResultsWe showed that expression PlncRNA-1, was significantly higher in CaP cells relative to normal prostate epithelial cells, as well as higher in human CaPs compared with normal tissues and benign prostatic hyperplasia (BPH). Silencing of PlncRNA-1 significantly reduced cell proliferation and induced apoptosis in CaP cell lines LNCaP and LNCaP-AI. Mechanistically, PlncRNA-1 suppression by siRNA resulted in a decrease of androgen receptor (AR) mRNA, protein and AR downstream target. Of note, blockade of AR signaling with siRNA also resulted in a suppression of PlncRNA-1 expression in CaP cell lines. ConclusionsOur study suggests reciprocal regulation of PlncRNA-1 and androgen receptor contribute to CaP pathogenesis and that PlncRNA-1 is a potential therapy target.

Chromosomal regions associated with prostate cancer risk localize to lamin B‐deficient microdomains and exhibit reduced gene transcription

The lamins are major determinants of nuclear shape and chromatin organization and these features are frequently altered in prostate cancer (CaP). Human CaP cell lines frequently have nuclear lobulations, which are enriched in A-type lamins but lack B-type lamins and have been defined as lamin B-deficient microdomains (LDMDs). LDMD frequency is correlated with CaP cell line aggressiveness and increased cell motility. In addition, LNCaP cells grown in the presence of dihydrotestosterone (DHT) show an increased frequency of LDMDs. The LDMDs are enriched in activated RNA polymerase II (Pol IIo) and androgen receptor (AR) and A-type lamins form an enlarged meshwork that appears to co-align with chromatin fibres and AR. Furthermore, fluorescence in situ hybridization and comparative genomic hybridization demonstrated that chromosomal regions associated with CaP susceptibility are preferentially localized to LDMDs. Surprisingly, these regions lack histone marks for transcript elongation and exhibit reduced BrU incorporation, suggesting that Pol II is stalled within LDMDs. Real-time PCR of genes near androgen response elements (AREs) was used to compare transcription between cells containing LDMDs and controls. Genes preferentially localized to LDMDs showed significantly decreased expression, while genes in the main nuclear body were largely unaffected. Furthermore, LDMDs were observed in human CaP tissue and the frequency was correlated with increased Gleason grade. These results imply that lamins are involved in chromatin organization and Pol II transcription, and provide insights into the development and progression of CaP.

First report of amphizoic amoebae isolated from edible Oyster mushroom- Pleurotus sajor-caju (Singer, 1949)

The present document serves as the first evidence that describes the presence of amphizoic amoebae in cap, stalk and internal tissues such as gills of the edible Oyster mushrooms, Pleurotus sajor-caju, packaged, and sold for public consumption. Samples tested over a 9 month period from different vegetable markets of Lucknow city revealed that out of 180 mushrooms, 176 (97.77%) yielded amphizoic amoebae, namely, Schizopyrenus, Naegleria, Hartmannella, Acanthamoeba and Vanella sp. Four samples of internal tissues yielded 6 strains of amoebae. A total of 17 Naegleria strains were isolated, out of which 5 were identified as Naegleria fowleri and were tested for their pathogenicity. The strains 2BNf01 and 5BNf04 were mildly pathogenic and strains, 4ANf02i, 4ANf03 and 8ANf05i were highly pathogenic. It was also observed that the most frequently observed species of amoebae belonged to the genus Acanthamoeba and a total 103 Acanthamoeba strains recorded.

Abstract A237: Elevated Mnk kinase activity is associated with malignant progression and reduced patient survival in human prostate cancer and can be therapeutically inhibited in human prostate cancer cells by the novel, orally bioavailable Mnk inhibitor cercosporamide.

Abstract In a wide variety of malignancies, progressive disease and reduced patient survival have been linked to increased function of eukaryotic translation initiation factor 4E (eIF4E), which selectively enhances the translation of potent growth and survival factors and oncoproteins (e.g. c-myc, VEGF, cyclin D1, Mcl-1, MMP-9, etc.). Recent published reports have shown that the oncogenic activity of eIF4E is critically dependent on the activity of the MNK kinases (Map kinase interacting kinase 1 and 2), which phosphorylate eIF4E at S209 (p4ES209). Increased p4ES209 has been linked to a variety of advanced cancers, notably head and neck and non-small cell lung cancers. Herein, we report that p4ES209 is significantly elevated in human prostate cancer (CaP) tissues (n=133) and associated with reduced patient survival. p4ES209 is elevated In both low grade (Gleason score ≤ 6) and high grade (Gleason score ≥ 7) CaP vs. adjacent normal prostate tissue (BPH) (p < 0.02 and p < 0.0001, respectively). Elevated p4ES209 in invasive CaP is related to significantly reduced patient survival (p = 0.0216) and increased risk of death from CaP (hazard ratio = 2.729). Moreover, p4ES209 is evident in the LNCaP, LNAI, CWR22Rv1 CaP cell lines, with levels being highest in the castrate-resistant cells (LNAI and CWR22Rv1). shRNA-mediated knock-down of MNK 1 and 2 reduced p4ES209 levels and specifically triggered apoptosis. Similarly, treatment of these cell lines with the novel, orally-bioavailable MNK inhibitor, cercosporamide, reduced p4ES209 in a dose-dependent manner and induced apoptosis coincident with reduced expression of key translationally controlled proteins (e.g. Mcl-1). Further, oral administration of this inhibitor can suppress p4ES209 in normal mouse tissues and in xenografted human cancers within 30 minutes, lasting at least 4 hours in tumor tissue. These data demonstrate the utility of cercosporamide in probing MNK function in vitro and in vivo and highlight the potential utility of targeting MNK for prostate cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A237.

Downregulation of Sec23A Protein by miRNA-375 in Prostate Carcinoma

Prostate carcinoma (CaP) is a leading cause of cancer-related death in men. We have previously determined the microRNA (miRNA) profile of primary CaP in comparison with nontumor prostate tissue. miRNAs are small, noncoding RNAs that inhibit protein synthesis on a posttranscriptional level by binding to the 3'-untranslated region (3'-UTR) of their target genes. In primary CaP tissue, we have previously found by miRNA sequencing that miR-375 and miR-200c were upregulated 9.1- and 4.5-fold, respectively. A computational analysis predicted the 3'-UTR of the SEC23A gene as a potential target for both miR-375 and miR-200c. Here, we show that the 3'-UTR of SEC23A mRNA is indeed a target for miR-375 and miR-200c and that both miRNAs downregulate Sec23A protein expression when ectopically expressed in human 293T cells. In primary samples of CaP, we found a direct correlation between reduction of SEC23A mRNA and overexpression of miR-375 but not of miR-200c. The reduced levels of Sec23A protein were inversely correlated to the increased amount of miR-375 in the LNCaP and DU145 CaP cell lines when compared with normal prostate fibroblasts. In primary CaP, we also detected decreased amounts of Sec23A protein when compared with corresponding normal prostate tissue. Ectopically overexpressed Sec23A in LNCaP and DU145 CaP cells significantly reduced the growth properties, indicating that Sec23A might play a role in the induction or growth of prostate carcinoma. Sec23A overexpression reduced cell growth but did not induce apoptosis, whereas inhibition of Sec23A stimulated cell proliferation.

Genome Organization and Transcription Response to Harvest of Two

Metallothioneins are a class of small cysteine-rich proteins that have been associated with increased tolerance to metal and oxidative stresses in animals, plants, and fungi. We investigated a metallothionein-like (mt-like) gene shown previously to be upregulated in fruiting bodies of the fungus Agaricus bisporus in response to post-harvest storage. Analysis of an A. bisporus genomic DNA cosmid library identified two similar mt-like genes (met1 and met2) arranged as a bidirectional gene pair transcribed from the same promoter region. The promoter contained regulatory elements including 9 metal responsive elements and a CAAT box region 220 bp downstream of met1 that showed striking similarity to a feature in Coprinopsis cinerea mt-like gene promoters. Transcriptional analysis showed that both met genes are significantly and rapidly (within 3 hours) upregulated during post-harvest storage and expression is significantly greater in stipe and cap tissues compared with the gills. However, a strong directionality of the promoter was demonstrated, as transcript levels of met1 were at least two orders of magnitude greater than those of met2 in all samples tested.

Expression and significance of matriptase and HAI-1 in prostate cancer

Objective To investigate the expression and significance of Matriptase and HAI-1 protein in prostate cancer (CaP). Methods Specimens of 46 prostate cancers,20 benign prostate hyperplasias (BPH),10 high-grade intraepithelial neoplasias (PIN),and 10 normal prostates (NP) were used. Expressions of Matriptase and HAI-1 proteins in specimens were detected by SP of immunohistochemistry. The results were analyzed in relation to the clinicopathological data. Results The protein levels of Matriptase in CaP tissues were significantly higher than PIN tissues(Z＝-2.150,P＝0.032),and the expression of matriptase in CaP and PIN was higher than that in BPH and NP (Z＝-3.270,P＝0.001;Z＝-2.817,P＝0.005). No statistically significant difference was observed between BPH and NP group (Z＝-0.895,P＝0.325). A progressive increase in the protein levels of Matriptase was observed with increasing tumor grade (rs＝0.583,P＜0.01) and clinical stages（rs＝0.611,P＜0.01）in CaP specimens. The protein levels of HAI-1 in BPH and NP tissues were significantly higher than CaP and PIN tissues（Z＝-3.277,-3.315,P＜0.01）,the levels of HAI-1 in PIN were higher than CaP (Z＝-2.310,P＝0.020). No statistically significant difference was found between BPH and NP (Z＝-0.872,P＝0.330). A progressive decrease in the protein levels of HAI-1 was observed with increasing tumor grades（rs＝-0.634,P＜0.01） and clinical stages（rs＝-0.521,P＜0.01）. The expressions of Matriptase and HAI-1 in CaP tissues showed negative correlations（rs＝-0.712,-0.560,-0.465,respectively,P＜0.01）. Conclusions The abnormal expressions of Matriptase and HAI-1 proteins may be important events during the progression of CaP in humans. Matriptase and HAI-1 Protein may be used as parameters for assessing the malignancy and prognosis of CaP. Key words: Serine endopeptidases/ME; Serine proteinase inhibitors/ME; Prostatic neoplasms/ME

Abstract 4173: Prostate derived Ets (E-twenty six) transcription factor (PDEF) as a prognostic biomarker for metastatic prostate cancer

Abstract While early stage prostate cancer (CaP) is unique in that many patients will take decades to progress to symptomatic disease; there exists a subset of patients whose disease may quickly become life-threatening. Unfortunately, due to the high incidence of CaP and clinicians’ inability to differentiate the majority of patients with disease that will never become symptomatic from the minority with aggressive life-threatening tumors, patients with CaP are often over-treated. Prognostic biomarkers, such as prostate specific antigen (PSA), have shown great efficacy in improving patients’ chances of surviving cancer by allowing physicians to identify the disease at an earlier stage, though there does not exist a biomarker for CaP that adequately differentiates disease that will quickly become metastatic. In order to facilitate prognosis, the expression of prostate derived Ets transcription factor (PDEF), a protein of significance to the progression of CaP, was studied. PDEF is expressed in normal epithelial cells and negatively regulates transcriptional networks controlling migration and invasion. Previous findings in breast cancer have also demonstrated that loss of PDEF expression results in upregulation of the antiapoptotic protein survivin, indicating a possible connection to growth and survival. Loss of PDEF expression has also been shown to increase the metastatic potential of several cancers. Patients PDEF expression was determined via immunohistochemistry staining of tissue microarray in set of over 700 archival CaP tissues taken at radical prostatectomy; and patients were subsequently stratified based on their tumor PDEF expression. The stratified CaP patients were then analyzed for the association of PDEF expression status with clinical outcomes. Univariate analysis showed that low PDEF expression is correlated with incidence of biochemical failure as defined by American Urological Association (AUA; OR=1.30, p=0.0493) or National Comprehensive Cancer Network (NCCN; OR=1.36, p=0.0164) definitions; and with metastatic disease incidence (OR=3.61, p=0.0047). Multivariate analysis also showed that low PDEF expression was associated with metastatic disease incidence (OR = 4.02, p=0.0146). Furthermore, Kaplan-Meier survival analysis showed that stratification of patients into subgroups based on PDEF expression (high, middle, low) was able to significantly predict relative time to biochemical failure (NCCN, p=0.0241) or metastatic disease (p=0.0084). This study demonstrates the potential usefulness of PDEF as a prognostic biomarker to differentiate those patients at the highest risk of developing life-threatening disease. We propose that the status of PDEF expression in CaP tissues could be used in conjunction with PSA to distinguish patients who are suitable for watchful waiting and patients who require aggressive treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4173. doi:10.1158/1538-7445.AM2011-4173

Abstract LB-308: Hypoxia affects gene expression and proteome of prostate cancer cells

Abstract Tumor-associated hypoxia facilitates malignant progression, metastasis and poor prognosis in prostate cancer (CaP); however, effects of oxygen tension (pO2) in CaP are just beginning to be investigated. To establish mechanisms whereby hypoxia enhances malignant properties and survival of CaP and to identify pO2-regulated tumor-associated macromolecules, we propagated CaP cell lines LnCaP, VCaP and DU145 in hypoxia (pO2=2 kPa) and compared them with normoxically grown cells (pO2=20 kPa); hypoxic cells proliferated faster and secreted more vascular endothelial growth factor (p<0.05). Transcriptome studies revealed different gene expression in cells grown in hypoxia relative to those in normoxia. Interestingly, in hypoxic cells transcripts associated with cancer and urologic disease were overexpressed in comparison to normoxic cells (p<0.001) suggesting an association of low pO2 and aggressive features of CaP. Further, we analyzed the transcriptome in primary CaP cells isolated by laser-capture microdissection (LCM) and whole CaP tissue. We compared the data to those from CaP cells cultured at pO2=20 kPa or pO2=2 kPa. Notably, hypoxia increased transcript levels for pyruvate dehydrogenase kinase isozyme 1, nuclear prelamin A recognition factor, glucose phosphate isomerase, and glyceraldehyde-3-phosphate dehydrogenase in all three cell lines (p<0.05) to levels comparable to those found in primary bulk tissue and LCM isolated cells (p<0.005). This finding suggests that gene expression in hypoxically cultured cells is more akin to that in tumor cells in situ than are cells grown normoxically. By 2D-gel electrophoresis, we found that change in pO2 affected the proteome mostly quantitatively (i.e., by change in spot intensity). Our results suggest that hypoxia affects transcriptome and proteome in CaP cells. In addition, we screened 20 patient sera and 20 healthy control sera for spontaneous antibodies cross-reactive with VCaP cells and found that the sera reacted with numerous proteins, some previously reported to elicit an immune response in CaP patients (e.g., nucleoporin 62 and transitional endoplasmic reticulum ATPase). By this method we detected numerous novel autoantigens (under validation). Currently we are clarifying the pO2 effects on the relationship of transcriptome, proteome and tumor-associated antigens in CaP cells. Support: DOD PC094680 (CRG), Minnesota Partnership for Biotechnology and Medical Genomics, Mayo Clinic Prostate SPORE 5P50CA091956 (FK); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-308. doi:10.1158/1538-7445.AM2011-LB-308

Abstract LB-384: Elevated eIF4E phosphorylation is associated with advanced malignancy and decreased patient survival and can be therapeutically inhibited by a novel, orally bio-available Mnk inhibitor

Abstract Malignant progression & reduced patient survival have been linked to increased function of eukaryotic translation initiation factor 4E (eIF4E), which selectively enables translation of potent growth/survival factors & oncoproteins (e.g. c-myc, VEGF, cyclin D1, Mcl-1). Recent reports have shown that the oncogenic activity of eIF4E is critically dependent on the activity of MNK kinases (Map kinase interacting kinases 1 and 2). Furthermore, increased eIF4E phosphorylation, a consequence of Mnk activity, has been linked to advanced cancers, including head & neck & non-small cell lung cancers. Herein, by immunohistochemical analysis of 133 prostatic tissues from 76 men, we report that eIF4E phosphorylation is significantly elevated in human prostate cancers (CaP) in relation to disease progression. eIF4E phosphorylation is associated with advanced disease (trend analysis, p < 0.0001) & is increased significantly in both low grade (Gleason score ≤ 6) & high grade (Gleason score ≥ 7) CaP vs. adjacent normal prostate tissue (BPH) (p < 0.02 and p < 0.0001, respectively). Elevated eIF4E phosphorylation in invasive prostate cancer CaP is related to significantly reduced patient survival (p = 0.0216) & increased risk of death from CaP (hazard ratio = 2.729). These data, along with data in experimental models implicating MNK activity in cellular transformation, indicate that therapeutic targeting of MNK activity may provide an attractive anti-cancer therapeutic strategy. Indeed, we now report the identification of a novel, small molecule Mnk inhibitor with IC50 values of 116 nm & 11nm against Mnk 1 & Mnk 2, respectively. This Mnk inhibitor suppresses eIF4E phosphorylation in a dose & time-dependent manner in a wide array of cancer cell lines, including melanomas, colorectal cancers, lymphomas, leukemias & prostate cancers. In HCT116 colon cancer, treatment with the Mnk inhibitor suppresses eIF4E phosphorylation and diminishes expression of the pro-survival factor Mcl-1. Mnk inhibitor treatment also suppresses cellular proliferation, induces apoptosis & dramatically diminishes soft agar colonization. Further, oral administration of this inhibitor suppresses eIF4E phosphorylation in normal mouse tissues and in xenografted human cancers within 30 minutes, lasting at least 4 hours in tumor tissue. Once daily (20mg/kg) or twice daily (10mg/kg) oral dosing significantly suppresses subcutaneous xenograft tumor growth. Moreover, oral dosing reduces dramatically the number and size of pulmonary metastases formed in mice injected intravenously with B16 melanoma cells & treated 12 consecutive days starting 1 day after cell injection. These data demonstrate the utility of this novel Mnk inhibitor in probing Mnk function in vitro & in vivo and highlight the potential anti-cancer therapeutic utility of suppressing Mnk function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-384. doi:10.1158/1538-7445.AM2011-LB-384

Abstract 4843: Genomic analysis of prostate cancer

Abstract Introduction: The most common male malignancy in the United States is prostate cancer; however its rate of occurrence varies significantly among ethnic groups. African American (AA) males have a 1.6 higher incidence rate for prostate cancer (CaP) and a 2.5 higher death rate as compared to Caucasian American (CA) Males in the USA. To date, no clear explanation is known for this glaring difference in CaP health disparity. A number of factors have been offered including genetic susceptibility, gene-environment interactions, presentation with late stage prostate cancer at diagnosis or presentation with more aggressive tumors, despite the stage and grade. We favor the hypothesis that altered gene expression plays a major role in the disparity observed in CaP incidence and mortality between AA/CA. Methods: We posed the question, what would be the result of Cap Tumor gene expression profiling if bioinformatics analysis of our existing cDNA microarray datasets did not focus on or stratify tumors based on race populations. Using data from our previously conducted micro array pilot project study which used AA and CA CaP tumors with a Gleason score of six and their matched controls;. We compared gene expression profiling in all non-tumor CaP tissue to all CaP tumors using the following filter conditions: minimal fold change of 2.0; t-test and Benjamin Hochberg. Results We identified thirty-one differentially expressed genes in tumors relative to non-tumor controls. Cystatin SN gene was down-regulated 13.19 fold in CaP tumors. Z-score analysis implicated the importance of multiple pathways: Toll-like receptor signaling, Colorectal cancer, MAPK signaling and Pathways in cancer. Genes common to some of the pathways included v-foes FBJ marine osteosarcoma viral oncogene, a key component of the activator protein-1 (AP-1) transcription factor or FOS; MAPK 13 and one DNA excision repair enzyme. MAPK13 (also known as p38δ) is a member of the p38 subfamily of Mitogen-activated protein kinase (MAPK). p38 MAPKs regulate diverse cellular responses including proliferation, differentiation, transformation, and programmed cell death. V-fos FBJ murine osteosarcoma viral oncogene homology (FOS) oncogene is a key component of the activator protein-1 (AP-1) transcription factor. Work is currently underway to determine if expression of either of the above genes is same for AA and CA males. To address this question we will use RT-PCR to determine expression in a panel of novel AA and CA prostate cancer cell lines. Conclusions: Results showed that the decision to not stratify tumors based on self-identified race when studying the genomics of prostate cancer revealed distinctly different genomic profile than those obtained when CaP tumors are stratified according to race. While a wealth of genetic information was uncovered in this study; the definitive next step will be to validate expression levels in AA and CA cell lines or tumor tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4843. doi:10.1158/1538-7445.AM2011-4843

619 EPIGENETIC CONTROL OF ANDROGEN ENRICHED LAMIN DEFINED MICRODOMAINS (LDMDS) IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011619 EPIGENETIC CONTROL OF ANDROGEN ENRICHED LAMIN DEFINED MICRODOMAINS (LDMDS) IN PROSTATE CANCER Brian T. Helfand, Yuanyuan Wang, Pekka Taiman, Katrin Pfleghaar, Robert D. Goldman, and Dale K. Shumaker Brian T. HelfandBrian T. Helfand Chicago, IL More articles by this author , Yuanyuan WangYuanyuan Wang Chicago, IL More articles by this author , Pekka TaimanPekka Taiman Chicago, IL More articles by this author , Katrin PfleghaarKatrin Pfleghaar Munich, Germany More articles by this author , Robert D. GoldmanRobert D. Goldman Chicago, IL More articles by this author , and Dale K. ShumakerDale K. Shumaker Chicago, IL More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1472AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Altered nuclear shape and DNA content have reproducibly been described in prostate cancer (CaP) specimens and are predictive of disease progression and metastasis. Nuclear lamin intermediate filaments are key determinants of nuclear size and shape and play a role in both DNA synthesis and transcription. We have shown that lamin-defined microdomains (LDMDs) are induced by knocking down lamin B1 (LB1) using shRNA. These LDMDs are enriched in euchromatin, accumulate activated RNA polymerase II (Pol IIo), are decreased in epigenetic histone marks for RNA elongation, and preferentially localize chromosome regions. The present study, 1) determined whether LDMDs are present within CaP cells and tissue and 2) characterized LDMD content. METHODS CaP tissue microarrays and human CaP cell lines with various invasive potential (DU-145, LNCaP, PC-3) were used. The presence and content of LDMDs were assayed using antibodies directed against lamin A/C, lamin B1, histone marks [AcH3 (euchromatin), H3K27me3 and H3K9me3 (heterochromatin), H3K36me3 (elongation phase of transcription)], Pol IIo and androgen receptor (AR). Transcription was assayed with BrUTP incorporation. RESULTS In CaP cells, LDMD presence appears to be correlated with disease aggressiveness and invasive potential. There was an increased frequency of LDMDs in high Gleason ≥8 disease vs. low grade (Gleason ≤6) disease (10.2% v. 2.1%; p<0.001). Based on immunostaining of epigenetic histone marks (AcH3, H3K27me3 and H3K9me3), LDMDs contain euchromatin which suggests a transcriptionally active microdomain. This is supported by the finding that LDMDs accumulate Pol IIo, and AR. Surprisingly, LDMDs are devoid of histone marks for the elongation phase of transcription and do not incorporate BrUTP. CONCLUSIONS LDMDs are present in CaP cells and their frequency appears to be correlated with tumor aggressiveness. LDMDs appear to be enriched for euchromatin. Although LDMDs accumulate AR and Pol IIo they appear to define a promoter proximal stalled state for Pol II. These studies will provide significant insights into the functions of LDMDs and will help to elucidate the mechanisms underlying the development and progression of aggressive CaP. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e249 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Brian T. Helfand Chicago, IL More articles by this author Yuanyuan Wang Chicago, IL More articles by this author Pekka Taiman Chicago, IL More articles by this author Katrin Pfleghaar Munich, Germany More articles by this author Robert D. Goldman Chicago, IL More articles by this author Dale K. Shumaker Chicago, IL More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

1607 HIGH-RESOLUTION DETECTION OF PTEN GENOMIC DELETION BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH) USING 330 FIXED PARAFFIN-EMBEDDED TISSUE SAMPLES

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 20111607 HIGH-RESOLUTION DETECTION OF PTEN GENOMIC DELETION BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH) USING 330 FIXED PARAFFIN-EMBEDDED TISSUE SAMPLES Jeremy Squire, Olga Ludkovski, Dave DeGrace, Andrew Evans, Kanishka Sircar, Tarek Bismar, and Maisa Yoshimoto Jeremy SquireJeremy Squire Kingston, Canada More articles by this author , Olga LudkovskiOlga Ludkovski Toronto, Canada More articles by this author , Dave DeGraceDave DeGrace Kingston, Canada More articles by this author , Andrew EvansAndrew Evans Toronto, Canada More articles by this author , Kanishka SircarKanishka Sircar Houston, TX More articles by this author , Tarek BismarTarek Bismar Calgary, Canada More articles by this author , and Maisa YoshimotoMaisa Yoshimoto Kingston, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1715AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Deletion of a tumour suppressor gene in routine formalin-fixed paraffin-embedded (FFPE) pathology sections is readily assayed by fluorescence in situ hybridization (FISH). However, the sectioning of the FFPE tissue samples often results in truncation artefacts of interphase nuclei leading to partitioning of the spherical shaped nucleus. As many as 20–40% of nuclei may be have been partially severed through the sectioning process leading to “false positive” signal losses and considerable interpretative difficulties. In this study a novel probe combination, for detecting gene deletions with high specificity was developed. METHODS Four color FISH probes were designed to detect genomic losses in the PTEN genomic region in 5 micron FFPE tissue sections. We evaluated the reliability of this assay using FFPE CaP tissue samples (tissue microarrays, n=330 patients), and mapping the breakpoint regions associated with PTEN deletions in this cohort derived from two institutions. RESULTS The probe combination selected reduced the rate of signal losses due to nuclear truncation by 50%. 132/330 (40%) of patient tumours were deleted for PTEN. The extent of deletion in each of the 132 tumors was mapped by FISH and the major class (41%) had an interstitial genomic deletion interval of 2Mb or less. The second most frequent class (28% of deleted tumors) involved genomic losses that were more heterogeneous in length, and involved PTEN deletions accompanied by adjacent genes on either side of the locus, with more losses extending in a telomeric direction. CONCLUSIONS FISH-based tumor suppressor deletion assay offers the advantage of highly specific and statistically significant quantitative evaluation of the gene locus of interest within individual cells in intact tissue. Consequently, CaP samples can be thoroughly characterized in an attempt to identify any PTEN heterogeneity, within or between foci in multifocal tumors. The clinical value of this assay is that presence of PTEN deletion by FISH is associated with earlier biochemical recurrence and increased mortality. For tumors with a homozygous deletion there is a strong association with hormone refractory CaP and metastatic disease. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e645 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jeremy Squire Kingston, Canada More articles by this author Olga Ludkovski Toronto, Canada More articles by this author Dave DeGrace Kingston, Canada More articles by this author Andrew Evans Toronto, Canada More articles by this author Kanishka Sircar Houston, TX More articles by this author Tarek Bismar Calgary, Canada More articles by this author Maisa Yoshimoto Kingston, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Evaluation and identification of markers of damage in mushrooms (Agaricus bisporus) postharvest using a GC/MS metabolic profiling approach

The aim of this research was to use the gas chromatography-mass spectrometry (GC/MS) profiling method coupled with chemometric tools to profile mechanically damaged and undamaged mushrooms during storage and to identify specific metabolites that may be used as markers of damage. Mushrooms grown under controlled conditions were bruise damaged by vibration to simulate damage during normal transportation. Three damage levels were evaluated; undamaged, damaged for 20 min and damaged for 40 min and two time levels studied; day zero and day one after storage at 4oC. Applying this technique over 100 metabolites were identified, quantified and compiled in a library. Random forest classification models were used to predict damage in mushrooms producing models with error rates of >10% using cap and stipe tissue. Fatty acids were found to be the most important group of metabolites for predicting damage in mushrooms. PLS models were also developed producing models with low error rates. With a view to exploring biosynthetic links between metabolites, a pairwise correlation analysis was performed for all polar and non-polar metabolites. The appearance of high correlation between linoleic acid and pentadecanoic acid in the non-polar phase of damaged mushrooms indicated the switching on of a metabolic pathway when a mushroom is damaged.

Regulation of seed germination in the close Arabidopsis relative Lepidium sativum: a global tissue-specific transcript analysis.

The completion of germination in Lepidium sativum and other endospermic seeds (e.g. Arabidopsis [Arabidopsis thaliana]) is regulated by two opposing forces, the growth potential of the radicle (RAD) and the resistance to this growth from the micropylar endosperm cap (CAP) surrounding it. We show by puncture force measurement that the CAP progressively weakens during germination, and we have conducted a time-course transcript analysis of RAD and CAP tissues throughout this process. We have also used specific inhibitors to investigate the importance of transcription, translation, and posttranslation levels of regulation of endosperm weakening in isolated CAPs. Although the impact of inhibiting translation is greater, both transcription and translation are required for the completion of endosperm weakening in the whole seed population. The majority of genes expressed during this process occur in both tissues, but where they are uniquely expressed, or significantly differentially expressed between tissues, this relates to the functions of the RAD as growing tissue and the CAP as a regulator of germination through weakening. More detailed analysis showed that putative orthologs of cell wall-remodeling genes are expressed in a complex manner during CAP weakening, suggesting distinct roles in the RAD and CAP. Expression patterns are also consistent with the CAP being a receptor for environmental signals influencing germination. Inhibitors of the aspartic, serine, and cysteine proteases reduced the number of isolated CAPs in which weakening developed, and inhibition of the 26S proteasome resulted in its complete cessation. This indicates that targeted protein degradation is a major control point for endosperm weakening.

Detection of Predictive Dynamics of Glucocorticoid Receptors in Xenopus Laevis Embryonic Tissues

The regulatory interactions in embryonic development that is guided by a complex set of spatially and temporally integrated stimuli result in the emergence of highly functional units. Since a better understanding of spatiotemporal responses provides a great insight to fundamental emergent patterns that may evolve from complex systems such as embryonic development, it is important to examine a generalized response to frequency-modulated stimulation from a system comprised of hundreds of cells where each cell has millions of interacting molecules. We present the dynamic responses of vertebrate embryonic tissues to time-varying localized chemical stimulation through a closed-loop microfluidic control that enabled localized spatiotemporal regulation of steroid hormone dexamethasone (DEX) in Animal Cap (AC) tissues isolated from gastrulating Xenopus embryos. We investigated dynamic cell-scale responses to precisely controlled stimulation by tracking the activity of a GFP-tagged Glucocorticoid Receptor (GR). Interestingly, the overall response had a predictive first-order behavior to periodic stimulation environments. We modeled these dynamic responses using first order differential equation with two different time derivatives: moving into and out of the nucleus. There was a good agreement between the predicted responses and the experimental results. Our approach provided a methodology for manipulating these biochemical inputs to examine and model the collective behavior of many biochemical reactions. The intricate cellular signaling and transport machinery responses were similar to behaviors in other complex systems, suggesting that even within a highly integrated and robust embryonic tissue, the overall system that we had studied converged toward a predictive first-order response. We believe that our results may suggest in the future that nanoscale molecular interactions in this developmental system results in highly regulated behavior, which we are able to determine through our feedback microfluidic control approaches in developmental biology.

Detection of Dynamic Spatiotemporal Response to Periodic Chemical Stimulation in a Xenopus Embryonic Tissue

Embryonic development is guided by a complex and integrated set of stimuli that results in collective system-wide organization that is both time and space regulated. These regulatory interactions result in the emergence of highly functional units, which are correlated to frequency-modulated stimulation profiles. We have determined the dynamic response of vertebrate embryonic tissues to highly controlled, time-varying localized chemical stimulation using a microfluidic system with feedback control. Our approach has enabled localized spatiotemporal manipulation of the steroid hormone dexamethasone (DEX) in Animal Cap (AC) tissues isolated from gastrulating Xenopus embryos. Using this approach we investigated cell-scale responses to precisely controlled stimulation by tracking the redistribution of a GFP-tagged DEX-reporter constructed from the human glucocorticoid receptor (GR). We exposed defined regions of a single AC explant to different stimulation conditions—continuous stimulation, periodic stimulation, and no stimulation. We observed collective behavior of the GR transport into the nucleus was first-order. Furthermore, the dynamic response was well-modeled by a first-order differential equation with a single time derivative. The model predicted that responses to periodic stimulations closely matched the results of the frequency-based experiments. We find that stimulation with localized bursts versus continuous stimulation can result in highly distinct responses. This finding is critical as controlled space and time exposure to growth factors is a hallmark of complex processes in embryonic development. These complex responses to cellular signaling and transport machinery were similar to emergent behaviors in other complex systems, suggesting that even within a complex embryonic tissue, the overall system can converge toward a predictive first-order response.