bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4897: Involvement of Akt isoforms in chemoresistance of carcinoma endometrial cells
Authors: Girouard Julie , Lafleur Marie-Judith , Leblanc Valérie , Asselin Eric
Published: August 2012

Akt (also known as PKB) is a crucial survival kinase that is normally activated by growth factors, but in tumors, it is constitutively activated at a high frequency through oncogenic events affecting its upstream regulation. Since studies have demonstrated that the three Akt isoforms exhibit distinct physiological functions, we propose that Akt isoforms may contribute differently in chemoresistance of endometrial carcinoma cells. In order to determine the specific role of each Akt isoform, we have stably produced transfectants in which the expression of Akt is downregulated by specific shRNAs for Akt1, Akt2 and Akt3 using the KLE endometrial carcinoma cell line. This endometrial cell line is known to constitutively express the three Akt isoforms and to highly resist to chemotherapeutic treatment. In this study, we have demonstrated that the downregulation of Akt1 and Akt2 by RNA interference strongly sensitizes KLE cells to cisplatin, doxorubicin and taxol and affects cell proliferation. Moreover, cisplatin treatment of these cells induced the activation of pro-apoptotic factors such as the cleavage of caspase -3, -6, -9 and PARP and it also inhibited the expression of anti-apoptotic factors such as XIAP and Bcl-2. Alternatively, we used constitutively active Akt expression vectors for each isoform to produce stable transfectants using the chemosensitive Hec 1A endometrial cancer cell line, in which no Akt activity is detected. Constitutive Akt1 and Akt2 expression leads to an increase in cell proliferation, as well as resistance to apoptosis after chemotherapeutic treatment in these stable transfectants. Moreover, Akt isoforms contribute differently in other cancer cell processes, such as migration. We showed that Akt2 blocks cell motility, while the activation of Akt1 or Akt3 does not show any effect on our endometrial cancer cell model. Our findings highlight the contribution of Akt1 and Akt2 isoforms in the molecular mechanisms that govern chemoresistance of endometrial carcinoma. Furthermore, Akt isoform-specific transfectants will provide a solid model to determine the involvement of each Akt isoform in tumor progression and metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4897. doi:1538-7445.AM2012-4897
Abstract 49: Targeting the TNF network in human ovarian cancer - A systems biology approach
Authors: Kulbe Hagen , Chakravarty Probir , Moore Robert , Iorio Francesco , Montoya Alexander , Saez-Rodriguez Julio , Cutillas Pedro , Balkwill Frances
Published: August 2012

In a recent publication (Kulbe et al Cancer Res 2011 epub ahead of print), we have shown how key pathways in cancer-related inflammation and Notch signaling are part of an autocrine malignant cell network in high-grade serous ovarian cancer, HGSOC. This network, that we have named the TNF network, has paracrine actions on angiogenesis, the stromal signature and the immune cell infiltrate in HGSOC. We have now used a systems biology approach, combining data from phospho-proteomic mass spectrometry and gene expression array analysis, to define the best therapeutic targets within the network and to identify drugs that may synergise with cytokine and chemokine inhibitors. First, we established a hierarchy of kinases involved in the TNF network and analyzed the constitutively active kinases in one of the high TNF network cell lines. Of 45 constitutively active kinases, 33 of these kinases showed direct interactions with each other. Next, we mapped gene expression microarray data onto the Connectivity Map of drugs in order to identify compounds having an effect on transcription similar to that of the TNF network. Among the identified candidate drugs were luteolin, apigenin and resveratrol. One of the known targets of this class of drugs is the protein kinase Casein kinase II (CSNK2A1), a kinase activated in association with the TNF network. In conclusion, we have identified kinases, particularly CK2, associated with the TNF network that may play a central role in sustaining the cytokine network and/or mediating its effects in ovarian cancer. We believe our findings have implications for our understanding of ovarian cancer biology and the development of new and more effective treatments for this disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 49. doi:1538-7445.AM2012-49
Abstract 50: Abrogation of stromal TGF-β signaling induces DNA damage and genetic/epigenetic alterations in neighboring epithelia
Authors: Achyut B. , Bader David A. , Pang Yanli , Harris Curtis C. , Yang Li
Published: August 2012

Inactivation of tumor suppressor genes in stromal fibroblast induces epithelial cancer development, suggesting an important role of stroma in epithelial homoeostasis. We investigated the underlying mechanisms in squamous cell carcinoma (SCC) development due to loss of TGF- signaling in FSP1+ fibroblasts (Tgfbr2fspKO). We found that stroma specific deletion of Tgfbr2 resulted a loss of p15 and p16, the cyclin dependent kinase (CDK) inhibitors in adjacent epithelia. In addition, there was an increased p21 methylation in the promoter region of the p53 binding site. These data suggest that multiple cell cycle checkpoints were compromised in the epithelia of Tgfbr2fspKO mice. Consistent with these observations, there was an increased cell proliferation in the epithelial compartment. The mechanisms mediating the cross talk between the epithelia and the stroma apparently involved inflammation that occurred only in the Tgfbr2fspKO mice but not in Tgfbr2flox/flox mice. This is characterized by an increased CD45+ leukocyte infiltration, increased cytokines expression (IL-10, IFN-γ TNF-α and GM-CSF) and elevated levels of iNOS, COX2 and NFκB. Interestingly, a significant DNA damage, featured by 8-Oxo-dG DNA adduct formation, and elevated expression of γ-H2AX, a histone protein in response to DNA double strand break, was observed in Tgfbr2fspKO mice. Inhibition of inflammation with COX2 inhibitor (Celecoxib) or germ free housing condition, significantly delayed epithelial hyperplasia/dysplasia and reduced DNA damage in Tgfbr2fspKO mice. Examination of human esophageal cancers showed a downregulation of TβR2 in the stromal fibroblast, and an increased inflammation and DNA damage similar to Tgfbr2fspKO mice. Together, our data demonstrate that attenuation of stromal TGF- signaling induces inflammation that in turn causes DNA damage, epigenetic and genetic alterations in epithelia. Therefore, therapeutic targeting of inflammation and tumor microenvironment may be useful in treating cancers with downregulation of TGF- signaling in stroma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 50. doi:1538-7445.AM2012-50
Abstract 5000: Epigenomic profiling of oral cancer
Authors: Yoshiba Sayaka
Published: August 2012

Epigenetic aberration, such as dysregulation of DNA mathylation is often associated with human carcinogenesis. To analyze DNA methylation profiles of oral cancers, and to identify the methylation pattern specific to the clinicopathological features, we performed genome-wide methylation analysis using Infinium HumanMethylation27 BeadArray system. We found 2,361 aberrantly methylated CpG sites in 59 oral cancers compared to 3 normal mucosal tissues. By unsupervised clustering analysis, oral cancer was divided into two methylation epigenotypes that are significantly correlated with incidence of lymph node metastasis and differentiation grades. We selected the classifier genes that can predict tumor metastasis in the training set, and demonstrated that they are validated to be predictive in the independent test sample, preoperative biopsy samples. In summary, we identified two methylation epigenotypes correlated with clinical outcome in oral cancer, and candidate classifier genes that would be useful in patient stratification for intensive therapy to overcome the tumor recurrence.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5000. doi:1538-7445.AM2012-5000
Abstract 5001: Epigenetic networks in breast cancer
Authors: Liu Chunmei , Dong Shoulian , Stevens Junko
Published: August 2012

Epigenetic mechanisms are essential for normal development and maintenance of normal gene expression patterns in humans. Recent studies suggest that epigenetic alterations may be the key initiating events in some forms of cancers and global changes in the epigenome are a hallmark of cancers. Phospholipase D (PLD) catalyzes the hydrolysis of phophatidylcholine to generate the lipid second messenger phosphatidic acid (PA) and choline and regulates multiple cellular pathways. Elevated expression of PLD1 promoted cell proliferation and has been associated with the progression and metastasis of multiple cancers. In this work, we showcased the applications of our new epigenetic tools by elucidating the changes in chromatin organization (3C), DNA methylation and microRNA expression that underlies the aberrant expression level of PLD1 in breast cancers. Differences in PLD1 expression and the epigenetic network between tamoxifen responsive (MCF-7) and non-responsive (MDA-MB-231) breast cancer cell lines were compared to associate epigenetic changes with their cancer phenotypes. Effects of DNA demethylation and histone acetylation induced by methyltransferase inhibitor (5-aza-2′-deoxycytidine) and histone deacetylase inhibitor (Trichostatin A) were also investigated to provide insights into the molecular mechanisms of epigenetic drugs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5001. doi:1538-7445.AM2012-5001
Abstract 5009: Identification of DNA methyltransferase target sites in cancer cells
Authors: Tiedemann Rochelle L. , Robertson Keith D.
Published: August 2012

Aberrant DNA methylation is commonly observed in cancer and is characterized by genome-wide hypomethylation and gene-specific hypermethylation, which is thought to contribute to genomic instability and tumor suppressor gene silencing, respectively. The DNA methyltransferases (DNMTs) are responsible for the establishment (DNMT3A, DNMT3B, DNMT3L) and maintenance (DNMT1) of DNA methylation patterns genome-wide. The mechanism by which DNA methylation patterns are altered in cancer is not well understood. Genome-wide unique and overlapping target sites for each of the DNMTs are also unknown both in normal and cancer states. Identification of DNMT target loci is essential in order to better understand how aberrant DNA methylation occurs in cancer. To study this process, DNMT mRNA levels were depleted both individually and in a combinatorial fashion via RNAi-based techniques in embryonic carcinoma cells. Following reduction in DNMT expression, the resulting DNA and RNA was analyzed for genome-wide DNA methylation and gene expression patterns, respectively. An affinity purification method, Methyl-Binding Domain (MBD)-seq, was used to capture and enrich methylated regions of the genome by utilizing the methyl-binding domain of MBD2b. The enriched methylated DNA was then used to construct an Illumina sequencing library. Global gene expression patterns were analyzed by microarray for each RNA sample. Next generation sequencing and microarray data were analyzed using several available algorithms (e.g. MACS) and high-throughput data software packages (e.g. Partek Genomics Suite) in order to construct and evaluate the DNA methylation profiles and subsequent gene expression changes that result from DNMT depletion. Our data show that distinct changes in DNA methylation profiles occur among the various DNMT knockdown samples that permit us to identify unique and cooperative target loci for each DNMT. Further elucidation of DNMT target sites holds great promise for enhancing our understanding of mechanisms that control aberrant DNA methylation that is observed in cancer as well as provide insight and rationale for targeting specific DNMTs in cancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5009. doi:1538-7445.AM2012-5009
Abstract 5020: Genetic and epigenetic analysis of tumors in a zebrafish model of melanoma
Authors: Casa Angelo , Bourque Caitlin , Zack Yael , Houvras Yariv
Published: August 2012

The activating BRAF kinase mutation V600E occurs in ∼60% of human nevi and melanomas. A transgenic strain of zebrafish expressing BRAF-V600E under the control of the mitfa promoter on a p53 mutant background develops melanomas with 100% penetrance. Previously, we showed that a transposon-based vector, MiniCoopR, allows us to program melanocytes to express a candidate gene in a specialized zebrafish strain Tg(mitfa:BRAFV600E); mitfa-/-; p53-/-. This strain lacks melanocytes, but the lineage can be rescued by MiniCoopR injection at the 1-cell stage. This genetic system allows us to create cohorts of transgenic animals that express candidate genes and assay the effects on tumor development. Previous work identified two histone methyltransferases (HMTs), SETDB1 and SUV39H1, which cooperate with BRAF-V600E to accelerate the onset and severity of melanoma development. A number of HMTs display alterations in expression level or copy number in human cancers compared to normal tissues, and we tested a panel of methyltransferases using the MiniCoopR system. We hypothesize that HMTs promote tumorigenesis by altering the epigenetic program of melanocytes. In order to explore the effects on melanocyte specification and differentiation, we overexpressed and depleted candidate HMTs in zebrafish embryos and performed in situ hybridization for lineage-specific markers, such as crestin, sox10, mitfa, dct, and tyr. HMTs that exert specific developmental phenotypes were further characterized by performing chromatin immunoprecipitation (ChIP). Our goal is to correlate the processes affected in the developing embryo with the epigenetic program to elucidate the mechanism by which HMTs influence tumorigenesis. Additionally, we are developing a strain of zebrafish Tg(mitfa:BRAFV600E); mitfa-/-; alb-/-; p53-/-; Tg(mitfa:GFP) in which melanocytes rescued with MiniCoopR express GFP. These rescued melanocytes can be programmed to express candidate HMTs and allow us to visualize melanocyte and melanoma development in vivo. The work presented here will help correlate cancer phenotypes with specific chromatin-modifying enzymes that may be the basis for new drug development and biomarker discovery.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5020. doi:1538-7445.AM2012-5020
Abstract 5035: MicroRNA expression profiles classify renal cell carcinoma subtypes
Authors: Wach Sven , Theil Anne , Stoehr Christine , Hartmann Arndt , Ekici Arif B. , Taubert Helge , Wullich Bernd
Published: August 2012

Background: Renal cell carcinoma (RCC) accounts for about 2-4% of all human malignancies. Hereby, the entity of papillary RCC (pRCC) is the second most common entity with about 10-15% of all RCC. According to histomorphological characteristics pRCCs can be further subdivided in two distinct subtypes. Type 2 pRCCs are correlated with a worse clinical outcome than type 1 pRCCs. In order to characterize these RCC subtypes we studied microRNA (miRNA) expression profiles of primary pRCCs type 1 and 2 as well as clear cell RCC (ccRCC). Material and Methods: We performed comparative miRNA expression profiling using Affymetrix miRNA microarrays. Ten to fifteen samples were analyzed for each subtype containing matched pairs of microdissected malignant and nonmalignant tissue. Hierarchical clustering was used to identify characteristic miRNA expression signatures. The expression of selected miRNAs was validated using quantitative PCR. Results: Hierarchical clustering was not able to identify miRNA expression signatures characteristic for all RCC subtypes, highlighting the heterogeneity among RCC tumors. But when stratified according to histological subtypes, tumor and nonmalignant tissue samples could be separated from each other. Interestingly, pRCC tumors of type 1 and 2 displayed a characteristic miRNA expression profile making it possible to separate these two tumor subtypes. A differential expression of miRNAs miR-200c, mir-143, miR-145, miR-210 and miR-10b could be confirmed by qRT-PCR in a cohort of pRCC samples (p<0.05; paired t-test). Applying a binary logistic regression model, a combination of only three of these miRNA miRNAs was sufficient to correctly determine the subtype of all pRCC tumor samples. Conclusion: Our findings demonstrate that miRNA expression in RCC is heterogeneous but there are characteristic miRNA expression profiles within RCC subtypes. In pRCC, specific miRNAs may reflect a molecular correlate to the histological differences between the two subtypes, which in turn are correlated to the clinical outcome.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5035. doi:1538-7445.AM2012-5035
Abstract 5040: Aberrant DNA methylation of microRNAs in hepatocellular carcinoma
Authors: Shen Jing , Wang Shuang , Zhang Yu-Jing , Kappil Maya , Wu Hui-Chen , Kibriya Muhammad G. , Wang Qiao , Jasmine Farzana , Ahsan Habib , Lee Po-Huang , Yu Ming-Whei , Chen Chien-Jen , Santella Regina M.
Published: August 2012

microRNAs (miRNAs) are a class of small non-coding RNAs involved in post-translational gene silencing by accelerating the degradation of mRNA and translational inhibition. miRNAs may regulate more than 60% of protein-coding genes’ translation. Previous studies have found that down-regulation of miRNAs is one of the most common features observed in solid tumors, including hepatocellular cancer (HCC). In the current study, we employed a genome-wide approach to test the hypothesis that methylation alteration in miRNA genes is an important mechanism causing deregulated miRNA expression in HCC. We analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using the HumanMethylation27 DNA Analysis BeadChips that include 255 CpG sites relevant to 64 miRNA genes. A volcano plot was used to display mean DNA methylation differences of all CpG sites. The hierarchical clustering for the significant CpG sites was performed to differentiate tumor and non-tumor tissues. After Bonferroni adjustment, a total of 54 CpG sites (27 relevant genes) significantly differed in methylation levels, with 53 CpG sites significantly hypermethylated in tumor compared to non-tumor tissues. Among the significant CpG sites, 15 sites have more than 2 fold tumor/non-tumor differences, 17 sites have a difference between tumor and non-tumor tissues of >10%, and 10 sites have both features. PANTHER ontology analysis suggested several enriched biological pathways for these miRNAs, including Wnt signaling, Ras, oxidative stress response, glutamine/glutamate conversion and mannose metabolism. The miRNAs methylation profiles were also compared by viral status (HBV/HCV) and AFB1 exposures. No statistically significant miRNA was identified. Analysis of a panel of candidate miRNAs (miR-196, miR-10a, miR-10b and miR-170) is ongoing in the same HCC tissues to evaluate whether the aberrant DNA methylation significantly correlates with expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5040. doi:1538-7445.AM2012-5040
Abstract 5184: Expression profiling of ECM proteins in advanced carcinoma of uterine cervix
Authors: Khan Rehan , Sharma Manoj , Sharma Alpana
Published: August 2012

Background Carcinoma of uterine cervix accounts for most common malignancy in Indian women. Migration is one of the important processes fundamental to cell invasion and dissemination. Role of extracellular matrix (ECM) proteins is also very important in this crucial process and cannot be ignored. ECM proteins are involved in cell migration, tissue remodeling, angiogenesis and cell adhesion. Fibulin-1 is cysteine-rich, calcium-binding extracellular matrix and plasma protein that has been implicated as playing a role in tumour progression by modulating cell morphology, growth, adhesion, and motility. Increased plasminogen activator (PA) secretion has been observed in malignant cells and tissue and PA is thought to be involved in the processes of tumorigenesis, cancer invasion and metastasis. Laminins are important for proliferation, differentiation, growth, migration & angiogenesis. Besides the structural function of nidogens, it has long been suggested that nidogens play a role in cell attachment and tumor angiogenesis. Objectives This study was aimed to investigate the expression and significance of ECM proteins (Fibulin-1, uPA, Laminin and Nidogen) in advanced carcinoma of uterine cervix. Material & Methods 40 patients of cancer cervix stage IIIb were included in this study. Circulatory levels of Fibulin-1, Laminin and Nidogen were measured using high sensitivity ELISA kits. uPA activity was measured by activity assay kit. The levels of biochemical markers were compared with 20 healthy females, taken as control. mRNA levels of these molecules were further assessed by Real Time RT-PCR. Data was analyzed statistically. Results Statistical significant (p < 0.001) increase was observed in Fibulin-1, uPA and Laminin levels and found to be significantly correlated with each other whereas Nidogen evels were insignificantly increased as compared to controls. Increased mRNA expression for all these molecules has also been observed in the isolated PBMC by qRT-PCR except nidogen (statistically insignificant increase). Conclusion The elevated serum/plasma levels and increased quantitative expression of these ECM proteins suggests their involvement in the pathogenesis of the disease. This study suggests that rise in ECM proteins might contribute to the complicated and unregulated process of metastasis in cancer of cervix. The association of extracellular matrix proteins and the prognosis of the disease may lead to have a better approach in treating the cervical cancer patients with a combination therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5184. doi:1538-7445.AM2012-5184
Abstract 5188: Altering cell attachment to growth substrate up-regulates growth differentiation factor 15 (GDF15) expression
Authors: Yong Koh Meng Aw , Zeng Yu , Vindivich Donald , Getzenberg Robert
Published: August 2012

Cell attachment to the extracellular environment is critical in determining cell fate. When normal cells lose attachment, they undergo anoikis. Cancer cells develop resistance to anoikis which results in metastasis. While there are many known genes alterations that can alter cell adhesion, less is known about how such a loss of cell adhesion may influence gene expression. We believe that a change in cell adhesion could trigger changes to gene expression, and the resulting changes play an important role in prostate cancer metastasis. Using prostate cancer cell lines, we examined gene expression changes in cancer cells brought about by loss of attachment to their growth substrate through chemical and physical means and how these changes play a role in prostate cancer. Microarray analysis was performed on LNCaP cells treated with latrunculin B, an actin cytoskeleton disrupting agent for 8 hours. Using quantitative real time PCR and immunoblot analysis, a member of the tumor growth factor beta superfamily, GDF15 was found to be consistently up-regulated when the cells were treated with either latrunculin B or other drugs that disrupt the actin cytoskeleton. These findings were observed not only in LNCaP but in PC3 and DU145 as well. GDF15 expression was rapidly increased in these three cell lines when cells were prevented from attaching to tissue culture plastic and the levels of GDF15 expression could be maintained as long as the cells were not allowed to re-attach. This up-regulation was reversed when cells were allowed to re-attach to growth substrate, or by regaining cell shape when the drugs were removed. GDF15 has been shown to be associated with metastatic prostate cancer as well as contributing to disease associated cachexia. In our study, we demonstrated that loss of cell adhesion was sufficient to increase GDF15 expression in a rapid and sustainable manner. This finding is novel and different from other reports that increased GDF15 led to altered cell shape and adhesion. We believe that our findings may provide a picture of the initial stages in metastasis when a cancer cell loses adhesion to the extracellular environment. Furthermore, our results reinforce the potential utility of GDF15 as a prognostic marker in prostate cancer, as well as the intricate relationship between the cell and the physical environment in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5188. doi:1538-7445.AM2012-5188
Abstract 5192: Platelet derived growth factor receptors differentially inform intertumoral and intratumoral heterogeneity
Authors: Kim Youngmi , Kim Eunhee , Guryanova Olga , Hitomi Masahiro , Sloan Andrew E. , Hjelmeland Anita B. , Rich Jeremy N.
Published: August 2012

Growth factor-mediated proliferation and self-renewal maintains tissue specific stem cells and is frequently dysregulated in cancers. Platelet-derived growth factor ligands and receptors (PDGFRs) are commonly overexpressed in gliomas and initiate tumors in genetically engineered models. PDGFRα alterations inform intertumoral heterogeneity towards a proneural glioma subgroup. We interrogated the role of PDGFRs in intratumoral heterogeneity with regards to differences in tumor cells. We found that PDGFRα is expressed only in a subset of gliomas while PDGFRβ is more commonly expressed in tumors but largely restricted to self-renewing tumorigenic glioma stem cells (GSCs). Targeting PDGFRβ genetically or with specific inhibitors attenuated GSC self-renewal, survival, tumor growth, and invasion. PDGFRβ targeting was associated with decreased activation of the cancer stem cell signaling node STAT3, and STAT3 activity rescued loss of PDGFRβ expression. These results demonstrate that a growth factor receptor family can differentially function to promote tumor heterogeneity. Our results may explain mixed clinical responses of anti-PDGFR based approaches, and suggest the need for integration of models of cancer as an organ system into development of cancer therapies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5192. doi:1538-7445.AM2012-5192
Abstract 5197: Quantitative phosphorproteomics identified GPER-initiated PKA signal transduction event in breast cancer stem cell
Authors: Lai Alan Chuan-Ying , Wang Yi-Ting , Lin Ruey-Jen , Wu Hsin-Yi , Chang Wen-Wei , Chang Wen-Ying , Chen Yu-Ju , Yu Alice L.
Published: August 2012

Human breast cancer stem cells (BCSC) identified recently have the ability to initiate neoplastic growth with differentiation and display higher resistance to chemotherapy and radiotherapy. The allure of targeting this specific group of cells to reduce mortality of disease has attracted research efforts toward BCSC. Although some unique properties of BCSC have been deciphered, the molecular mechanisms are still poorly understood. Here, we applied in-house developed techniques to quantitatively compare the phosphoproteomic profiles between BCSC and non-cancer stem cells (nonCSC) derived from a xenograft of human breast cancer. For quantitative analysis, we utilized a SEMI-strategy for multiplexed label-free quantitation combining pH/acid-controlled IMAC chromatography and LC-MS/MS for relative quantitation of site-specific phosphorylation. This strategy effectively increases the phosphopeptide quantitation coverage. The differentially expressed phosphoproteins were mapped to multiple signaling pathways including NOTCH, CDK/ERK and JAK-STAT pathways, which potentially orchestrate the self renewal and stemness of BCSC. We also demonstrated ∼2 fold greater expression of GPER in BCSC than non-BCSC population, and GPER signaling could induce PKA-mediated phosphorylation of BAD in BCSC. Silencing of GPER via RNAi or mutation of phosphorylation sites of BAD led to reduced proliferation to 40.8 and 53.2 % of control, respectively, and the RNAi silencing of GPER decreased the mammosphere formation of BCSC to 43.5 % of control. These findings implies the importance of GPER and its downstream PKA pathway to the maintenance of stemness characteristics of BCSC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5197. doi:1538-7445.AM2012-5197
Abstract 5201: Suppression of WNT1 expression inhibits the maintenance and expansion of cancer stem cells in a mouse model of breast cancer
Authors: Kim Ran-Ju , Choi A-Ram , Roh Kyung-Jin , Kim Soo-Rim , Park Ju-Hyun , Lee Su-Youn , Park Se-Jin , Nam Jeong-Seok
Published: August 2012

Cancer relapse is the most common cause of mortality in breast cancer patients. The cancer stem cell (CSC) hypothesis proposes that CSCs are the root of cancer. CSC-targeted therapies may prevent cancer relapse and provide more effective treatment. It has been known that stem cells can be enriched in non-adherent sphere culture. To identify molecular targets in breast CSC, we compared transcription levels of stem cell-related genes between tumorspheres established from Balb/c mice-derived 4T1 mammary carcinoma and mammospheres from the mammary glands of Balb/c mice. The most differentially expressed gene was found to be Wingless-type MMTV integration site family, member 1 (WNT1). To determine whether WNT1 has any relevance in breast carcinoma, we analyzed the expression of WNT1 in the human matched normal breast and breast cancer samples. Immunofluorescent staining showed that WNT1 expression was elevated ∼17-63-fold in cancerous tissues compared with non-cancerous tissues from the same breast cancer patients. Functionally, knockdown of WNT1 in 4T1 cells reduced the Aldefluorpos cell compartment enriched in CSCs and suppressed the properties of CSCs, including anti-apoptosis, sphere-forming ability, and tumor initiation ability in vivo. Collectively, these results show that WNT1 expression gives rise to properties of CSC in breast cancer. Therefore, the targeting of WNT1-associated signaling may provide an effective therapeutic approach in the treatment of advanced breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5201. doi:1538-7445.AM2012-5201
Abstract 5206: Systems biology identification of processes/pathways facilitating the development of high-grade serous ovarian cancer
Authors: Balch Curt , Jadhav Rohit , Fang Fang , Pilrose Jay , Li Meng , Shen Changyu , Li Lang , Huang Tim H-M , Nephew Kenneth P.
Published: August 2012

Previously, based on well-known stem cell phenotypes (sphere formation, potent tumorigenicity, embryonic gene expression, and chemoresistance), we used patient tumors to isolate high-grade serous (HGS) ovarian cancer-initiating cells (OCICs) that express the cell surface markers CD44 (a hyaluronate receptor) and CD117 (c-kit) (Zhang et al., 2008, Cancer Res 68:4311). Here, to identify biological pathways that underlie HGS ovarian tumorigenesis, including the specific role of OCICs in that process, we performed integrated analyses of global gene expression, DNA copy number, and DNA methylation patterns. To that end, OCICs and “bulk” (i.e., disaggregated but unsorted) tumor cells were compared to primary human normal ovarian surface epithelial (nOSE) cells that, along with fallopian tube epithelial (FTE) cells, represent hypothesized cells-of-origin for HGS pelvic cancers. Principle components analysis and unsupervised hierarchical clustering revealed distinct DNA methylation and gene expression profiles for replicate (n=3) samples of each cell type (i.e., OCIC, bulk tumor, and nOSE). Gene ontology and additional unsupervised analyses of “gene expression signatures” corresponding to the epithelial-to-mesenchymal transition (82 of 159 signature genes), TGF-beta (47 of 86 genes), and Wnt (75 of 151 genes) pathways demonstrated well-defined segregation of the three cell types, with mesenchymal and Wnt signaling hierarchies being nOSE > OCIC > bulk tumor cells. To examine early events in HGS ovarian tumorigenesis, previously published (Tone et al., 2008, Clin Cancer Res 14:4067) gene expression datasets were employed to show that nOSE, OCIC, and bulk ovarian tumor cell transcriptomes distinctly segregate from normal (FTE) and malignant fallopian epithelial transcriptomes. Interestingly, the largest divergence was between the nOSE and FTE gene expression profiles. Finally, DNA methylation and gene expression patterns were integrated, implicating specific networks/circuits in ovarian carcinogenesis. Taken together, these results support a mesenchymal-to-epithelial transition during ovarian tumorigenesis, reveal legitimate therapeutic targets (at the pathway level), and raise uncertainty that surface epithelial cells of the fallopian tube and ovary are reciprocal cells-of-origin for HGS pelvic carcinomas.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5206. doi:1538-7445.AM2012-5206
Abstract 5232: Tyrosine kinase inhibitor, gefitinib targets ZAP-70 over expressing chronic lymphocytic leukemia cells
Authors: Gibson Spencer , Xiao WenYan , Yoon Ju Yoon , Noh Edward , Brown Michelle , Johnston James B.
Published: August 2012

Most patients with chronic lymphocytic leukemia (CLL) initially respond to chemotherapy but relapse. There is a subset of CLL patients that have aggressive disease characterized by an over expression of the tyrosine kinase ZAP-70. These ZAP-70+ patients have a poorer survival and are more resistant to chemotherapy. The biological effects of ZAP-70 in CLL are believed to be related to its ability to enhance activation of Syk with subsequent triggering of down-stream modulators, such as ERK and AKT. Normally, Syk is activated by increased phosphorylation by Lyn, which is increased in CLL, or by activation of the B-cell receptor. Gefitinib is a tyrosine kinase inhibitor that is presently used to treat lung cancer. The drug inhibits epidermal growth factor receptor (EGFR) tyrosine kinase activity but has activity against >20 other kinase targets, including members of the Src family of kinases, such as Lyn and Syk. We thus evaluated the cytotoxic activity of gefitinib against primary CLL cells using the MTT assay. Gefitinib was cytotoxic to ZAP-70+ (≥20% cells positive) CLL samples (median IC50, ∼3.0 µM) while ZAP-70- patients failed to respond to gefitinib with a median IC50 of >15.0 µM. Jurkat cells, which are T cells that express high levels of ZAP-70, were more sensitive to gefitinib than B cell lines that did not express ZAP-70 (BJAB and NALM6). Further studies confirmed that gefitinib was inducing cell death through apoptosis. Gefitinib was also effective against ZAP-70+ CLL cells that were resistant to fludarabine and chlorambucil indicating a novel mechanism of action to standard chemotherapy. Enhanced apoptosis was observed in ZAP70+ CLL and Jurkat cells exposed to gefitinib and fludarabine or chlorambucil, but this was not observed in ZAP-70- or the B cell lines. In contrast, another EGFR inhibitor, erlotinib, had no effect against ZAP-70+ CLL cells indicating that gefitinib was inhibiting a kinase unaffected by erlotinib. When Jurakat and ZAP-70+ CLL cells were treated with gefitinib, there was reduced total tyrosine phosphorylation and decreased tyrosine phosphorylation of ZAP-70, and Syk but no difference in Lyn phosphorylation. Furthermore, gefitinib blocked B cell receptor activation and mediated cell survival. Taken together, these results indicate that gefitinib may be useful agent in the treatment of ZAP-70+ CLL, either alone or in combination with fludarabine or chlorambucil. Ongoing studies are determining the precise mechanism of action of gefitinib in ZAP-70+ CLL and assessing its effects on the CLL microenvironment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5232. doi:1538-7445.AM2012-5232
Abstract 5237: Interrogating cell heterogeneity and dormancy in prostate cancer disseminated tumor cells
Authors: Ruppender Nazanin S. , Brown Lisha , Coleman Roger , Lakely Bryce , Coleman Ilsa , Welty Christopher , Gulati Roman , Nelson Peter S. , Lange Paul H. , Corey Eva , Vessella Robert , Morrissey Colm
Published: August 2012

Dissemination of tumor cells to the bone is an early event in prostate cancer (PCa) and is often insufficient by itself for the development of metastases; while PCa cells may be present in bone marrow, not all patients develop tumors in the bone. Therefore, the characterization of disseminated tumor cells (DTC) in bone marrow is important. We hypothesize that profiling gene expression in DTC could identify molecular cues that determine why some DTC are dormant, which dormant cells have the potential to reactivate, and which DTC will remain dormant. Our objectives were: (1) to develop a method to profile the transcriptome of individual PCa cells, (2) use the method to profile tumor cell heterogeneity and markers of dormancy in individual DTC from patients, and (3) use LuCaP xenograft lines to examine tumor cell heterogeneity and dormancy in vitro. We have demonstrated previously that we can obtain 15,441 positive probes from one PCa cell using a high stringency cut off on Agilent 44K oligonucleotide arrays. Furthermore, using the same high stringency cut off, the sensitivity and specificity between 1 and 10 cells was 0.640 and 0.946 respectively, demonstrating a very low false positive rate. Using this methodology, we are comparing the gene expression profile of 10 individual DTC from each of 5 patients with no evidence of disease four years after radical prostatectomy and 10 individual DTC from each of 5 patients with advanced metastatic disease. Additionally, our laboratory has developed 24 LuCaP PCa xenografts that do not proliferate in typical in vitro cell culture conditions. However, we have developed a technique using reactive bone marrow stroma that allows for in vitro culture and proliferation of these xenografts. Thus, we are comparing individual LuCaP cells in three cellular ‘states’ in vitro (1) dormant, (2) activated, and (3) inactivated to interrogate tumor cell heterogeneity and identify markers of tumor cell dormancy. Using this new technique for in vitro culture of xenografts, we show that cell contact, not only to the underlying stroma but also to other tumor cells, is required for proliferation of LuCaP cells in vitro. Furthermore, we show that proliferating, “active” LuCaP cells can be induced to enter a dormant, “inactive” state through lack of cell-cell contact. In conclusion, our studies will address the heterogeneity of single DTC isolated from the bone marrow of patients with PCa, and using both clinical and experimental specimens, identify potential genes associated with dormancy in PCa.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5237. doi:1538-7445.AM2012-5237
Abstract 5246: Use of γH2aX and other biomarkers to quantify distribution of drug activity in solid tumors
Authors: Saggar Jasdeep K. , tannock ian F.
Published: August 2012

Background: The disorganized vasculature within solid tumors leads to decreased drug delivery and poor penetration of drugs from blood vessels to the tumor cells. Our laboratory has used quantitative immunohistochemistry (IHC) to show limited intratumoral distribution of the auto-fluorescent drugs doxorubicin and mitoxantrone. Most anti-cancer drugs are not fluorescent and do not have antibodies that recognize them; there is a need for biomarkers that can be used to quantify their distribution and efficacy in different regions of solid tumors. The aim of the present study was to characterize the spatial distribution within solid tumors of biomarkers of drug effect that can be used to test drug efficacy. Methods: Cleaved caspase 3 (a marker of apoptosis), γH2aX (a marker of DNA damage) and Ki67 (a marker of cell proliferation) were quantified in tumor sections in relation to blood vessels (recognized by CD31) using monoclonal antibodies and IHC. To validate the use of these biomarkers we compared their time-dependent distribution with (i) the distribution of auto-fluorescent doxorubicin and (ii) the distribution of a fluorescent monoclonal antibody that detects melphalan-induced DNA adducts. The biomarkers were then used to study the distribution of docetaxel in relation to tumor blood vessels. Results: The distributions of doxorubicin in MCF7 & MDA-MB-231 xenografts and of melphalan-induced DNA adducts in MCF-7 & EMT-6 tumors decreased exponentially with increasing distance from tumor blood vessels; they were similar to the distribution of γH2aX at 10 minutes after injection of these drugs. The distributions of cleaved caspase 3 and of change in Ki-67 at 24 hours following treatment also mirrored the distribution of the drugs. The distribution of these biomarkers following injection of docetaxel also showed a sharp decrease in drug activity with increasing distance from tumor blood vessels in solid tumors (MCF-7, PC-3 & A431). Conclusions: Activation of γH2aX occurs within 10 minutes after injection of several drugs and is the preferred biomarker for studying distribution of drug activity in tumors. There is heterogeneity of activity of many anticancer drugs in solid tumors, with rapid decrease in activity with increasing distance from tumor blood vessels. Poor distribution of drugs to cells within tumors is an important cause of drug resistance. Supported by a research grant from the Canadian Institutes of Health Research

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5246. doi:1538-7445.AM2012-5246
Abstract 5318: DPC4 loss results in the activation of alternative oncogenic pathways in pancreatic cancer
Authors: Brosnan Jacqueline A. , White Catherine M. , Iacobuzio-Donahue Christine A.
Published: August 2012

Introduction: Pancreatic cancer remains one of the most lethal forms of cancer, with a five-year survival rate of less than five percent. Many of these patients succumb to complications from metastasis. We have shown that genetic inactivation of DPC4, a major factor in TGF-β signaling, is significantly correlated with widespread metastasis in pancreatic cancer patients at autopsy. We hypothesize that DPC4 loss may be driving pancreatic cancer metastasis by forcing the TGF-β signal down an alternative pathway. Methods: DPC4 was stably restored into three cell lines that harbor a homozygous deletion of the locus (A2.1, A6L, and BxPC3), forming isogenic pairs of DPC4−/− and DPC4+ cells. Matched DPC4−/− and DPC4+ cells were treated with TGF-β1 for 24 hours prior to protein extraction. Immunoblotting was performed for total and phospho Akt (Pi3K pathway), p44-42 (ERK pathway), and Stat3 (JAK-Stat pathway), as well as nuclear and cytoplasmic p65 (NFκB pathway), as these networks have previously been identified as being activated by TGF-β. Immunohistochemistry was performed on 50 surgically resected pancreatic cancers for pAkt. Results: DPC4 was successfully introduced into DPC4−/− cells, as evidenced by semi-quantitative PCR for DPC4, as well as a TGF-β luciferase assay. DPC4+ cells displayed characteristic differences in cell behavior, such as decreased proliferation, migration, and invasion, compared to DPC4−/− cells. Western blot analysis of Smad-independent TGF-β pathways in three sets of DPC4-complemented pancreatic cancer cell lines revealed patterns of preferential activation of the Pi3K and ERK signaling pathways in cells lacking DPC4. In surgically-resected pancreatic cancers, activation of Pi3K was associated with the presence of lymph node metastases (p=0.03), while concomitant DPC4 loss and Pi3K pathway activity was associated with poor survival in pancreatic cancer patients (p=0.02). Conclusions: Restoration of DPC4 into DPC4−/− cells results in the abrogation of Pi3K and ERK signaling, and simultaneous DPC4 loss and Pi3K activation correlates to a poor prognosis of pancreatic cancer patients at surgery. Further studies will confirm whether genotype-specific activation of the ERK and Pi3K pathways plays a role in pancreatic cancer cell behavior, particularly those processes that directly impact metastasis (i.e. migration and invasion). Nevertheless, it is intriguing to consider the Pi3K and ERK pathways as therapeutic targets in pancreatic cancer patients with loss of DPC4.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5318. doi:1538-7445.AM2012-5318