bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 522: The epithelial-mesenchymal transition-related transcription factor ZEB suppresses Claudin expression in breast cancer cells.
Authors: Arima Yoshimi , Hosonaga Mari , Saya Hideyuki
Published: August 2014

Triple-negative breast cancer (TNBC) is classified as a heterogeneous group, because TNBCs are defined as breast cancer negative for the estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2 (HER2). We show that TNBC cell lines can be divided into those which express the repressor of transcription Zinc finger E-box binding homeobox 1 (ZEB1) and the tight junction protein claudin-1. ZEB1 has been identified as a key transcription factor eliciting epithelial-mesenchymal transition (EMT) and cancer progression. We found that knockdowns of ZEB1 in TNBC cells induce an epithelial-like morphology and induce the mRNA expression of claudin-1.> The claudin family is the principal constituent of tight junctions in epithelial cells. We also found that ZEB1 binds to a claudin-1 promoter sequence by DNA-mediated pull-down assay. Furthermore, in breast cancer specimens, we observed a reciprocal expression of ZEB1 and claudin-1. These findings suggest that ZEB1 represses claudin-1 expressions in TNBCs. We then analyzed the association of clinical outcomes with ZEB1 and claudin-1 expressions by using a publicly available gene expression database of breast cancer patients. The overall survival rates in the category which tested positive for ZEB1 and claudin-1 is poor. To further characterize the effects of ZEB1-positive breast cancer cells on this phenotype of cancer, we investigated tumor formations following the co-inoculation of ZEB1-expressing MDA-MB-231 cells and claudin-1-expressing HCC1937 cells into nude mice. Although HCC1937 cells alone didn't form tumors in our orthotopic xenograft models, tumor formations were observed when the cells were mixed with MDA-MB-231 cells. These data suggest that the ZEB1-expressing breast cancer cells do promote tumorigenicity of claudin-1-positive cancer cells. We hypothesized that the heterogeneity on gene expressions within the tumor tissue is associated with malignancy and therapeutic resistance of breast cancers.

Citation Format: Yoshimi Arima, Mari Hosonaga, Hideyuki Saya. The epithelial-mesenchymal transition-related transcription factor ZEB suppresses Claudin expression in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 522. doi:10.1158/1538-7445.AM2013-522
Abstract 5225: Single-cell gene-expression programs and basal-like breast cancer.
Authors: Wang Chun-Chao , Jamal Leen , Bajikar Sameer S. , Atkins Kristen A. , Janes Kevin A.
Published: August 2014

Basal-like carcinoma is a subtype of breast cancer that is characterized by poor prognosis and high intratumor heterogeneity. In basal-like breast epithelia, we have identified two anticorrelated gene-expression programs that arise among single extracellular matrix (ECM)-attached cells during organotypic 3D culture. The first program contains multiple TGFβ-related genes including TGFBR3, and its heterogeneous induction is critical to suppress ductal branching. The second program contains JUND together with the basal-like marker, KRT5. Homogenizing JUND expression in single cells leads to 3D acini with bridged lumina that are similar to cribiform ductal carcinoma in situ. TGFBR3 and JUND together comprise a circuit that is interconnected via four negative-feedback loops. Computational modeling of the circuit predicts damped, antiphase oscillations upon stimulation with endogenous impulses of TGFβ-like ligand, and we directly observe these spontaneous responses in 3D culture by live-cell imaging. The TGFBR3-JUND circuit is remarkably conserved in early basal-like tumors that heterogeneously express KRT5, suggesting that asynchronous circuit dynamics are active in this patient subset. We further show that the circuit is strongly dependent on ECM engagement, as detachment leads to a rewiring that is triggered by RPS6 dephosphorylation and maintained by juxtacrine signaling from tenascin C. Breast tumor heterogeneity need not stem from partial basal-like differentiation and could instead reflect dynamic toggling of individual cells between expression states.

Citation Format: Chun-Chao Wang, Leen Jamal, Sameer S. Bajikar, Kristen A. Atkins, Kevin A. Janes. Single-cell gene-expression programs and basal-like breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5225. doi:10.1158/1538-7445.AM2013-5225
Abstract 5226: Understanding single cell variability in oncogene induced pathway aberration in lung cancer.
Authors: Deb Dhruba , Rajaram Satwik , Larsen Jill , Minna John , Wu Lani , Altschuler Steven
Published: August 2014

Progression of normal epithelial cells towards malignancy is marked by alteration in signaling pathways involved in cellular growth and development. However, at any given stage of oncogenic progression, it is unknown to what extent different signaling pathways are altered and whether the degree of signaling change is similar from cell to cell. A quantitative survey of signaling alterations that are associated with common oncogenic changes will allow us to connect the widely observed signaling heterogeneity in cancer to the genetic changes leading to its disease states.

Here, we study an isogenic series of human bronchial epithelial cells (HBECs) that have been oncogenically transformed with the sequential addition of common lung cancer mutations (p53null, p53null+KRASV12, p53null+KRASV12+c-myc). To quantify the degree of signaling alteration, we performed immuno-fluorescence assays and acquired images of HBECs stained with signaling markers representing important lung cancer pathways including WNT, JAK/STAT, TGFβ, IFNγ, MAPK, AKT and TNFα pathways. We extracted intensity-based features from individual cells using our in-house image analysis platform. This single-cell analysis revealed a high degree of signaling change within each of our HBEC populations, suggesting that signaling alteration can be different from cell to cell and from pathway to pathway.

We further quantified the degree of pathway alteration along our sequential oncogenic manipulations by using standard machine learning techniques. Our analysis revealed significant changes in the WNT, TGFβ and MAPK pathways upon oncogenic progression. Loss of p53 alone caused a major shift in TGFβ signaling; addition of mutant KRASV12 altered the MAPK, TNFα and JAK/STAT pathways; Addition of a third genetic manipulation, C-MYC, induced dramatic shifts in the WNT and IFNγ pathways. We are currently in the process of inferring crosstalk among multiple pathways in HBECs and a panel of ten lung cancer cell lines. Taken together, our results suggest that the degree of signaling alteration across diverse signaling pathways can be correlated with a specific oncogenic state in lung cancer.

Citation Format: Dhruba Deb, Satwik Rajaram, Jill Larsen, John Minna, Lani Wu, Steven Altschuler. Understanding single cell variability in oncogene induced pathway aberration in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5226. doi:10.1158/1538-7445.AM2013-5226
Abstract 5227: Quantitative network models of signaling and drug response in melanoma.
Authors: Korkut Anil , Wang Weiqing , Molinelli Evan , Miller Martin , Kaushik Poorvi , Aksoy Arman , Sander Chris
Published: August 2014

Use of targeted drug combinations are potentially key to preventing emergence of resistance to initially successful single agents such as RAF inhibitors. Thus, systematic nomination of novel drug combinations is important to develop effective therapeutic strategies. Here, we present an experimental-computational network pharmacology method to infer quantitative signaling networks in tumor cells, predict response to perturbations, and ultimately, nominate targeted drug combinations that will generate a desired phenotypic response. We use a series of targeted drugs, singly or in combination to perturb cancer cells. We measure quantitative proteomic (total and phoshpo-protein levels) and phenotypic (e.g. cell viability, apoptosis, cell cycle progression) response profiles to perturbations. Proteomic responses are measured with the reverse phase protein array technology. Next, we infer quantitative network models, using the response profiles as training sets. Solution space of all network model configurations is prohibitively large and Monte Carlo based inference algorithms fail to generate accurate network models in sizes relevant for cancer biology. In order to solve the inference problem, we have adapted an iterative and probabilistic inference algorithm, belief propagation from statistical physics. Resulting network models are based on simple non-linear differential equations and quantitatively link signaling events to phenotypic changes. We have generated quantitative network models of signaling in RAF inhibitor resistant melanoma cell lines in sizes (i.e. ∼100 nodes) unreachable by other network inference algorithms. We have nominated novel combination therapies through combinatorial in silico perturbations of all nodes in derived networks and experimentally tested the predicted phenotypic responses.

Citation Format: Anil Korkut, Weiqing Wang, Evan Molinelli, Martin Miller, Poorvi Kaushik, Arman Aksoy, Chris Sander. Quantitative network models of signaling and drug response in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5227. doi:10.1158/1538-7445.AM2013-5227
Abstract 5231: Highly conserved ceRNA regulatory interactions cooperate with genomic variability to modulate drivers of tumorigenesis.
Authors: Chiu Hua-Sheng , Yang Xuerui , Martínez María Rodríguez , Iyer Archana , Sumazin Pavel , Califano Andrea
Published: August 2014

MicroRNAs (miRs) have been repeatedly linked to tumorigenesis and tumor progression, suggesting their potential value as biomarkers and targets for therapeutic intervention. Recent evidence suggests that mRNAs compete for binding and regulation by a finite pool of miRs, thus regulating each other as competitive endogenous RNAs (ceRNAs). The extent of this new regulatory layer and its potential to alter the expression of cancer genes has been shown in several tumor types, but the actual impact of ceRNA interactions on disease remains unclear.

In this study, using TCGA (The Cancer Genome Atlas) tumor datasets, we provide evidence that ceRNA play an important role in tumor etiology. Leveraging millions of shRNA-mediated perturbations from LINCS (Library of Integrated Network-based Cellular Signatures), we extensively validated ceRNA interactions in breast and prostate cancer. Moreover, we identified over 160,000 ceRNA interactions that are conserved across tumor types despite being implemented by miR programs with tumor type-specific expression. We present evidence that these ultraconserved interactions are mechanistically responsible for the dysregulation of the majority of oncogenes and tumor suppressors through TCGA-profiled genomic alteration of their cognate ceRNA regulators. Using targeted biochemical validation in cell lines, we verified the potential of ceRNA regulators to target ESR1, an established oncogene and drug target in breast cancer, and APC, an established tumor suppressor in colon adenocarcinoma.

Our analysis suggests that ceRNA interactions are implemented through competition for multiple miRNAs, rendering them virtually independent of single miRNA kinetics and availability, and more likely to be conserved across contexts. Using TCGA samples, we demonstrated that conserved ceRNA interactions enable systematic identification of genomic alterations that contribute to aberrant expression of hundreds of cancer genes in each of eight distinct tumor contexts. We submit this network as a resource for analyzing ceRNA regulation in datasets where reverse engineering context-specific networks is infeasible due to low sample number or lack of miRNA expression profiles.

Citation Format: Hua-Sheng Chiu, Xuerui Yang, María Rodríguez Martínez, Archana Iyer, Pavel Sumazin, Andrea Califano. Highly conserved ceRNA regulatory interactions cooperate with genomic variability to modulate drivers of tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5231. doi:10.1158/1538-7445.AM2013-5231
Abstract 5232: Salivary proteome as an in vivo model to study breast cancer progression.
Authors: Streckfus Charles F.
Published: August 2014

Background: To date, because of the complexity and heterogeneity of breast cancer, no individual model recapitulates all aspects of this disease. Since both the mammary and salivary glands are exocrine glands and that the literature has shown cross-talk between the two glands, we hypothesize that, secondary to breast cancer, the malignancy's rapid growth alters the proteomic content of the tissue microenvironment and that these changes may manifest in up or down regulation of salivary protein concentrations, which can be used as a sentinel body fluid for breast cancer modeling. Verification that salivary proteomic content can be used to model breast cancer progression employing HER2 biology would provide strong support for immediate translational testing in clinical trial settings that would further be accelerated with the use of saliva-based testing as one of the most easily accessible body fluids.

Objective: The objective of this study was to compare the salivary protein profiles of pooled saliva specimens from individuals diagnosed with ductal carcinoma of the breast with and without HER2/neu overexpression as a potential alternate to current HER2/neu assessment methodologies.

Methods: Pooled (n=10 subjects/pooled specimen) stimulated whole saliva specimens from women were analyzed. One pooled specimen was from healthy women, another was from women diagnosed with Stage IIa (T2N0M0) invasive ductal carcinoma (IDC) without positive HER2/neu receptor status. A third pooled specimen was from women diagnosed with Stage IIa (T2N0M0) IDC with a positive HER2/neu receptor status. Isotopically tagging proteins in the tumor groups and comparing them to the healthy control group measured differential expression of proteins. Experimentally, saliva from each of the pooled samples was trypsinized and the peptide digests labeled with the appropriate iTRAQ reagent. Labeled peptides from each of the digests were combined and analyzed by reverse phase (C18) capillary chromatography on an LC-MS/MS mass spectrometer equipped with an LC-Packings HPLC. Results: The results of the analyses yielded approximately 174 differentially expressed proteins in the saliva specimens. There were 20 proteins unique to HER2/neu positive receptor status and 28 proteins that were unique to HER2/neu negative status. Conclusions: The study suggests that saliva is a fluid suffused with solubilized by-products of oncogenic expression and that these proteins may be differently expressed in the presence of HER2/neu positive receptor status. It also provides support to the novel idea of using salivary secretions to study breast cancer progression

Citation Format: Charles F. Streckfus. Salivary proteome as an in vivo model to study breast cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5232. doi:10.1158/1538-7445.AM2013-5232
Abstract 5233: Modeling ionizing radiation exposure in vitro and in vivo using the Virtual Tumour.
Authors: Fernandez Eric , Orrell David , Brightman Frances , Fell David , Chassagnole Christophe
Published: August 2014

Ionizing radiation exposure induces DNA double strand breaks in mammalian cells. In response, cells activate repair mechanisms which involve cell cycle arrest at discrete transition points of the cell cycle. The results in an accumulation of cells in S and G2 phase in an irradiated cell population over a period of time following exposure.

Using the Virtual Tumour, a cell population simulator of cancer cells and tissue, we have built a model of irradiation of tumour cell culture in vitro as well as tumour tissue in vivo for various cancer cell types. We show how cell cycle timings and radiosensitivity can accurately reproduce both in vivo and in vitro response to irradiation-induced DNA damage. We also study how the radiosensitization effect of gemcitabine is time-dependent. This demonstrates how the Virtual Tumour can help maximise therapeutic efficacy of combinations involving irradiation.

Citation Format: Eric Fernandez, David Orrell, Frances Brightman, David Fell, Christophe Chassagnole. Modeling ionizing radiation exposure in vitro and in vivo using the Virtual Tumour. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5233. doi:10.1158/1538-7445.AM2013-5233
Abstract 5240: Differential anticancer drug response by high-dimensional protein monitoring.
Authors: Nishizuka Satoshi S. , Ishida Kazushige , Wakabayashi Go
Published: August 2014

Cellular signal transduction responses to drugs are not always identical; rather, they depend on concentration, administration method, and time after administration. To monitor drug-induced signaling, we produced “reverse-phase” protein arrays (RPPAs) using cell lysates from three anticancer drugs (5FU, CIS, CPT), two administration methods (sustained, Sus; temporal, Tem), and three concentrations (High, Medium, Low) of drug administration over a 24-hour period. The combination of drug types, concentrations, and administration methods collectively yielded 18 unique drug administration conditions. Each RPPA was individually probed with 37 primary antibodies from our antibody panel; these antibodies recognize the proteins involved in genotoxic stress response. The quantitative readout was then analyzed to see if there was: (i) a general trend in protein dynamics in response to each drug condition; and (ii) significance in those proteins that exhibited similar dynamics. We first classified 666 time-course protein dynamics. About 38% of the protein dynamics showed an increasing trend, whereas about 32% showed a decreasing trend. Proteins exhibited earlier peaks when stimulated by Low or Medium concentrations for all drugs, while proteins exhibited later peaks when stimulated by High concentrations. Clustering analysis of protein dynamics induced by drugs revealed that each dynamic class included proteins of diverse functions. Hence, it is unlikely for cells to respond to particular drugs in a defined pattern. Subsequently, to clarify whether any functional associations are prioritized in drug responses, we evaluated the similarities between the protein dynamics by distance correlation (DC, which is known to have a higher value the tighter the association), to see if any pair of protein dynamics demonstrated a geometric similarity associated with its known interaction. It appeared that apoptosis-related proteins tended to show high DC to other proteins when anticancer drugs were administrated at high enough concentrations to induce apoptosis. We also found that structural cell proteins, such as keratins, showed high DC to apoptosis-related proteins, and confirmed that when these two functional protein groups interact, what results is degradation in the progression of drug-induced apoptosis.These observations indicate that high-dimensional drug response protein dynamic monitoring by RPPAs may offer a unique opportunity to find drug-specific general dynamic trends and potentially novel protein interactions unique to drug administrative conditions such as dose.

Citation Format: Satoshi S. Nishizuka, Kazushige Ishida, Go Wakabayashi. Differential anticancer drug response by high-dimensional protein monitoring. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5240. doi:10.1158/1538-7445.AM2013-5240
Abstract 5241: Identifying signaling pathways associated with medulloblastoma subtypes from "omic" data.
Authors: Archer Tenley C. , Weeraratne Shyamal Dilhan , Francois Jessica Pierre , Pomeroy Scott L.
Published: August 2014

Medulloblastoma, the most common malignant pediatric brain tumor, comprises four subtypes classified by their distinct molecular signatures. Wnt and SHH subtypes up-regulate those respective signaling pathways, while Group 3 and Group 4 tumors are less understood and have the worst prognosis. Treatment is currently limited to surgical resection, radiation and broad-spectrum cytotoxic chemotherapy drugs that frequently devastate cognition in survivors. In order to develop patient-specific targeted therapies we need to understand the underlying molecular events that drive the tumor in each patient. Previously, our research group and others sequenced whole exomes from a total 92 primary tumors from patients in comparison to their matched normal blood. Over 1,700 mutated genes were identified. The overwhelming majority of mutations occurred only once in the entire cohort, and just 12 genes were recurrently mutated with statistical significance. Statistically significant mutated genes include genes involved in subtype-specific signaling events, such Wnt and SHH which have been shown to be causative in those subtypes, and others are involved in chromatin and epigenetic regulation. The diversity of mutations makes it difficult to resolve which mutations drive oncogenesis, especially in Groups 3 and 4. Most patients in these subtypes do not harbor a mutation in one of the 12 significantly mutated genes, leaving an open question as to why these patients developed cancer. The aim of this study is to employ systems biology to interweave singleton genes into networks to identify candidate drives in each patient and ultimately resolve the underlying common pathways that caused each cancer. We have identified singleton mutations in patients that substitute for the statically significant mutated genes. Modeling individual patient level data onto networks predicts novel driver events in each patient. These candidate driver events are then functionally validated in vitro. In addition to mutation, oncogenic events can occur via copy number changes, chromatin regulation, transcription regulation, mRNA processing, or protein degradation. We hypothesize that common pathways are targeted in multiple tumors of the same type, despite the obvious differences in the patterns of somatic mutations.

Citation Format: Tenley C. Archer, Shyamal Dilhan Weeraratne, Jessica Pierre Francois, Scott L. Pomeroy. Identifying signaling pathways associated with medulloblastoma subtypes from "omic" data. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5241. doi:10.1158/1538-7445.AM2013-5241
Abstract 5245: C-Kit isoforms predict melanoma drug sensitivity.
Authors: Phung Bengt , Steingrímsson Eiríkur , Rönnstrand Lars
Published: August 2014

The receptor tyrosine kinase c-Kit is an essential regulator of melanocyte and melanoma development. Alternative splicing of c-Kit produces one long variant, c-Kit(+), with a tetrapeptide GNNK located in the extracellular juxtamembrane domain, and one short variant, c-Kit(-), which lacks this sequence.

In this study we characterize for the first time the function of the GNNK tetrapeptide sequence in melanoma. Our results show that step by step elimination of the GNNK tetrapeptide gradually increases receptor tyrosine phosphorylation, ubiquitination and downstream MAP kinase ERK activation. Successively increasing insert length, progressively leads to reduced melanoma cell survival during dacarbazine or rapamycin drug treatment. Our results indicate that the ratio between c-Kit isoforms might be useful as a prognostic marker for melanoma drug response.

Citation Format: Bengt Phung, Eiríkur Steingrímsson, Lars Rönnstrand. C-Kit isoforms predict melanoma drug sensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5245. doi:10.1158/1538-7445.AM2013-5245
Abstract 5247: Human melanoma cells resist oxidative stress due to overactive MEK/ERK and AKT/mTOR pathways and overproduction of melanin.
Authors: Calianese David , Cammarata Garrett , Brum Gabriella , Szulak Kevin , Best Charles , Higgins Katelyn , Wan Yinsheng
Published: August 2014

Melanoma is considered as one of the most aggressive and less treatable cancers. While various models of mechanisms have been proposed, the transformation from tranquil melanocytes to invasive melanoma cells remains an enigma. Alteration of cellular redox status has been suggested to be associated with cellular transformation. We compared primary human melanocytes and melanoma cells and their responses to oxidative stress. Both cells were treated with H2O2 at doses between 5 and 250μM. Microscopic data showed that melanoma cells are more resistant to H2O2 treatment. Western blot and confocal microscopic data showed that in melanoma cells, H2O2 does not induce CRT translocation which is considered as a mechanism through which melanocytes surrender to oxidative stress and induces immunogenic cell death. Furthermore, Western blot analysis showed that melanoma cells express EGFR, responsive to EGF treatment and have constitutive activation of AKT and mTOR, which are all related to better survival. In contrast, in the primary human melanocytes, EGFR expression is almost undetectable. And confocal microscopic data confirm this observation. Treatments of cells with MEK/ERK inhibitor U0126 and PI3K/AKT inhibitor LY294002 attenuates S6 phosphorylation. We also observed that in melanoma cells, but not in melanocytes, low dose (5 to 250μM) of H2O2 induces melanin production that is recognized to protect from further oxidative stress. Collectively, our data suggest that active MEK/ERK and AKT/mTOR pathways and up-regulation of melanin production contribute to the resistance of melanoma cells to oxidative stress and attribute to the aggressiveness of melanoma. Our data provides insights into the understanding of the molecular mechanisms of the transformation from melanocytes to melanoma and support the notion from the most recently published proteomic studies that EGFR remains one of the most important targets for better clinical management of melanoma.

Citation Format: David Calianese, Garrett Cammarata, Gabriella Brum, Kevin Szulak, Charles Best, Katelyn Higgins, Yinsheng Wan. Human melanoma cells resist oxidative stress due to overactive MEK/ERK and AKT/mTOR pathways and overproduction of melanin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5247. doi:10.1158/1538-7445.AM2013-5247

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 526: Combined inhibition of mTOR and AKT activity induces potent apoptosis in epithelial ovarian cancer cells .
Authors: Hussain Azhar R. , Uddin Shahab , Ahmed Maqbool , Al-Kuraya Khawla S.
Published: August 2014

Mammalian target of rapamycin (mTOR) and Phosphatidylinositol 3-kinase (PI3K) are two key components of the PI3K/Akt/mTOR signaling pathway. These two signal transduction cascades regulate a number of physiological cell functions including differentiation, proliferation, apoptosis and autophagy. Dysregulation of these pathways have been found in many cancers including DLBCL, thyroid cancer, breast cancer, prostate cancer and lung cancer. mTOR has been shown to activate the PI3-kinase/AKT survival pathway and understanding the interplay among signaling molecules in the PI3K/Akt/mTOR pathway is of utmost importance. Two distinct mTOR complexes; mTORC1 and mTORC2, have been identified. mTORC1 is downstream of Akt, sensitive to rapamycin inhibition, and controls cap-dependent protein translation. In contrast, mTORC2 is directly upstream of Akt and is resistant to rapamycin. In this study, we investigated the effect of mTOR inhibition on cell viability and induction of apoptosis in epithelial ovarian cancer (EOC) cells, either alone or in combination with PI3-kinase/AKT inhibitor. We found that Torin2, an mTOR inhibitor, decreased cell viability and induced apoptosis in EOC cell lines in a dose dependent manner. Torin2 also inactivated mTOR1 and mTOR2 as well as downstream targets of mTOR; 4E-BP1 and p70S6. Torin2 treatment of EOC cell lines also induced mitochondrial dependent apoptosis via activation and cleavage of caspases. Finally, combination treatment of EOC cells with Torin2 and LY294002, a PI3-kinase/AKT inhibitor induced a synergistic apoptotic response via inactivation of AKT, mTOR1 and mTOR2 and activation of caspases. These studies will give us an insight on the molecular relationship between these survival pathways and suggest that simultaneous inhibition of mTOR and PI3-kinase/AKT pathways may be beneficial for the management of EOC.

Citation Format: Azhar R. Hussain, Shahab Uddin, Maqbool Ahmed, Khawla S. Al-Kuraya. Combined inhibition of mTOR and AKT activity induces potent apoptosis in epithelial ovarian cancer cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 526. doi:10.1158/1538-7445.AM2013-526
Abstract 5265: Akt regulates insulin-like growth factor binding protein-3 by modulating its expression in human non-small cell lung cancer cells.
Authors: Jin Quanri , Min Hye-Young , Whang Young Mi , Kim Yeul Hong , Lee Ho-Young
Published: August 2014

Since it has been suggested the insulin-like growth factor (IGF)-independent antitumor effect of insulin-like growth factor binding protein-3 (IGFBP-3) in human cancer cells, the mechanism underlying the regulation of IGFBP-3 in the cells has not been fully studied. Therefore, in this study, we investigated the precise role of Akt in the regulation of cellular IGFBP-3 in human lung cancer cells. Inhibition of PI3K/Akt by siRNA transfection targeting Akt or by treatment with an inhibitor (LY294002) enhanced antiproliferative and proapoptotic effects of IGFBP-3 in lung cancer cells. Additional pharmacological (by using a small molecule inhibitor) or genomic (by siRNA-based silencing of PTEN or overexpression of dominant-negative Akt) approaches suggest that Akt regulates the expression and stability of IGFBP-3 protein. Further characterization of the role of Akt in the regulation of IGFBP-3 by introduction of constitutively active form of each Akt subtype indicates that Akt3 destabilized cellular IGFBP-3 and suppressed retinoic acid-induced IGFBP-3 expression in both TSC2-/- MEF and H226B lung cancer cells. Collectively, these data suggest a novel function of Akt, especially Akt3, as a negative regulator of IGFBP-3 and, further, the possible implication of combined treatment targeting IGFBP-3 and Akt in the treatment of lung cancer.

Citation Format: Quanri Jin, Hye-Young Min, Young Mi Whang, Yeul Hong Kim, Ho-Young Lee. Akt regulates insulin-like growth factor binding protein-3 by modulating its expression in human non-small cell lung cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5265. doi:10.1158/1538-7445.AM2013-5265
Abstract 5267: STAT signaling is totally deregulated in malignant pleural mesothelioma: What are the reasons.
Authors: Arzt Lisa , Quehenberger Franz , Halbwedl Iris , Popper Helmut H.
Published: August 2014

Malignant pleural mesothelioma (MPM) is an aggressive tumor caused by former asbestos exposure with a mean overall survival of 12 months from diagnosis. The knowledge about predictive factors of outcome is poor and MPM is resistant to conventional therapy. The long latency period in MPM pathogenesis as well as the absence of early symptoms is responsible for the late diagnosis of this disease.

From our recent study and published data we know that the regular rapid progressive type of MPM uses several signaling pathways regulating cell growth and survival. Asbestos causes genetic modifications, most notably the upregulation of cell survival and growth signaling pathways as well as the expression of other proteins that favor the resistance of MPM to apoptosis and chemotherapy. In order to develop more effective therapy than the currently used chemotherapy, novel targets have to be identified in MPM.

The JAK/STAT signaling pathway is the principal signaling mechanism for lots of cytokines or growth factors in mammalians. JAK activation induces a variety of biological responses like cell proliferation, differentiation, cell migration, and apoptosis.

Our investigations demonstrate that the STAT signaling pathway is totally deregulated: STAT1 is upregulated, whereas STAT3 is downregulated. SOCS1 and SOCS3 - which represent a negative feedback loop and which are regulators of STAT1 and STAT3 - are totally missing in MPM. Investigations in six mesothelioma cell lines showed that STAT1 is highly expressed and that SOCS1 is missing whereas the expression of pSTAT1 (Tyr701) can be stimulated and therefore be increased by Interferon-gamma treatment. The expression levels of other components of the STAT signaling pathway in these cell lines are currently under investigation.

It is known that protein expression is controlled by noncoding RNAs, e.g. microRNAs (miRNA) and we investigated the possible impact of miRNAs on the STAT signaling pathway in MPM. We identified miR-30d* as a regulator of phosphorylated STAT1 (Ser727). Furthermore we found that miR-19b, miR-30b, miR-30c and miR-222 are upregulated which are predicted to target SOCS1 and SOCS3 - this could be an explanation for the missing expression of these proteins. MiR-21 is extremely upregulated in MPM, a miRNA that is associated with a wide variety of human tumors and which targets STAT3.

Understanding the role of STATs in MPM could be the first step into the development of a targeted therapy for these tumors.

Citation Format: Lisa Arzt, Franz Quehenberger, Iris Halbwedl, Helmut H. Popper. STAT signaling is totally deregulated in malignant pleural mesothelioma: What are the reasons. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5267. doi:10.1158/1538-7445.AM2013-5267
Abstract 5273: Systematic functional screening, miRNA and target expression analyses reveal miR-19, -124, -583, -129 and -876 as key nodes in prostate cancer proliferation networks.
Authors: Östling Päivi , Leivonen Suvi-Katri , Kohonen Pekka , Aakula Anna , Hintsanen Petteri , Aittokallio Tero , Perälä Merja , Kallioniemi Olli
Published: August 2014

To discover microRNA (miRNA) regulation of pathways relevant for proliferation of prostate cancer cells we performed gain-of-function screening with 1129 miRNA molecules in five prostate cancer (PCa) cell lines. Using proliferation, viability and apoptosis as read out we identified 25 miRNAs increasing and 48 miRNAs decreasing cell viability (threshold FDR q-value 3000 genes containing the predicted targets (TargetScan) and performed gene set enrichment analysis (MSigDB v. 2.07). Positively enriched gene sets (total of 17, q-value <0.01) contained signatures such as mitosis and androgen signalling and the most enriched gene was AURKA. The negatively enriched gene sets (total of 207, q-value <0.01) involved Ras-signaling and focal adhesion and the most down regulated gene was CAV1. Network visualization of the core target genes identified five miRNAs (hsa-miR-19a, −32, -124a, -583 and −876–3p) predicted to regulate both the positively and negatively enriched target genes suggesting a central role for both these miRNAs and the core targets in PCa. Our systematic screening approach combined with in vivo expression, gene set enrichment analyses and network visualization provide an unbiased way of revealing novel miRNAs -core targets important in PCa.

Citation Format: Päivi Östling, Suvi-Katri Leivonen, Pekka Kohonen, Anna Aakula, Petteri Hintsanen, Tero Aittokallio, Merja Perälä, Olli Kallioniemi. Systematic functional screening, miRNA and target expression analyses reveal miR-19, -124, -583, -129 and -876 as key nodes in prostate cancer proliferation networks. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5273. doi:10.1158/1538-7445.AM2013-5273
Abstract 5274: Expression of selected miRNA and miRNA processing genes in oral precancer and cancer tissues.
Authors: Roy Bidyut , Roy Roshni , De Sarkar Navonil , Paul Ranjan Rashni
Published: August 2014

Introduction: Oral leukoplakia is a premalignant lesion that long has been considered to confer increased risk for the development of Oral Cancer which is highly prevalent in India. Oral lichen planus (OLP) on the other hand is a chronic inflammatory disease whose malignant potential is debatable. Since not all oral precancerous lesions progress to oral cancer, molecular markers like miRNA expression could be used to diagnose and predict progression of disease that cannot be detected histopathologically.

Objective: Study of intra- and inter-variation in the expression of a few microRNAs (26a1, 423, 34b, 29a, 193a, 137 and 205) and microRNA processing (XPO5, GEMIN3 and RAN) genes in the three disease types, namely, oral cancer, leukoplakia and lichen planus.

Methodology: Total RNA was extracted from paired disease and normal tissues from 15 oral cancer, 19 leukoplakia and 19 OLP patients. Expression of microRNAs and microRNA processing genes were studied using TaqMan assay. Subsequent statistical analyses were performed for interpretation of expression data.

Results: Expression of mir 26a-1 and mir 137 genes was dysregulated >2 folds in 70% of the cancer samples while expression of remaining microRNAs and mir-processing genes were deregulated in a few samples. In leukoplakia, expression of mir-26a-1 was dysregulated >2 folds in 50% of the samples while dysregulation in expression of the remaining miRNAs was observed in 1/3rd of the patients. In OLP, there was an interesting trend of unidirectional up or down regulation in the expression of mir26a-1, mir29a, mir-34b and mir-137 genes. We also observed that the average expression of miRNA genes differed among the three patient groups.

Conclusion: Deregulation in microRNA expression could not be correlated with individual's tobacco habits. Variation in expression of microRNA as well as miRNA processing genes was observed within individuals of the same patient group (i.e. cancer or leukoplakia or lichen planus) and between any two patients groups. There was correlation in expression of microRNAs which probably explains the unidirectional increase or decrease of miR expression in OLP. Finally, the mean expression level in the three types of disease tissues differed significantly. But expression of more miRNA genes in more samples would be necessary for molecular markers.

Citation Format: Bidyut Roy, Roshni Roy, Navonil De Sarkar, Ranjan Rashni Paul. Expression of selected miRNA and miRNA processing genes in oral precancer and cancer tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5274. doi:10.1158/1538-7445.AM2013-5274
Abstract 5285: miRNA expression pattern for predicting prognosis of ovarian serous carcinoma.
Authors: Cui Shiying , Zhang Xinchen , Yu Xiaotang , Ding Yanfang , Zhao Jinyao , Zhao Henan , Wang Chang
Published: August 2014

Objective: One of the best prognosis predictors for patients with epithelial ovarian cancer is clinic pathological stage at diagnosis. The goal of this study is to develop and validate a gene expression profile that can predict prognosis to ovarian serous carcinoma (OSC) at early and advanced stages.

Methods/materials: To identify a unique miRNAs pattern that involved in the progression of OSC at earlier and later stages, miRNA microarray was performed by using 38 China tumor-bank specimens with OSC at stage I and III in a retrospective analysis. The expression of dysregulated miRNAs were validated using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in external cohort OSC at stage I and stage III. Kaplan-Meier analysis was further performed to analysis the correlation between the expression of key microRNAs and prognosis.

Results: Of the 768 miRNAs analyzed from microarray, 26 miRNAs were significantly either up or down-regulated with 2 fold difference at least in OSC at stage I compared to III. qRT-PCR results confirmed that 19 miRNAs were significantly down-regulated and 7 was up-regulated in OSC at stage III compared to stage I, which is consistent with the results of microarray. Downregulation of 5 miRNAs was significantly associated with poor disease-specific survival (P<0.05). The patient survival rate of miRNAs high-expression group was 77.8% higher than the low-expression group 33.3%. The different expression patterns of key miRNAs in OSC at stage I versus III were further validated by in situ hybridization.

Conclusions: Our results suggest that miRNA plays an important role in the progression of OSC and 5 miRNAs may be considered as the novel candidates of clinic biomarkers for predicting OSC outcome.

Citation Format: Shiying Cui, Xinchen Zhang, Xiaotang Yu, Yanfang Ding, Jinyao Zhao, Henan Zhao, Chang Wang. miRNA expression pattern for predicting prognosis of ovarian serous carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5285. doi:10.1158/1538-7445.AM2013-5285
Abstract 5286: A genetic selection system for microRNA targets identification and validation.
Authors: Zhou Nanjiang , Mo Yin-Yuan
Published: August 2014

Overwhelming evidence has indicated that microRNAs can play a fundamental role in regulation of diverse cellular functions and dysregulation of microRNA expression could lead to a variety of disorders including human cancer. Although significant progress has been made in the past years in discovery of microRNAs and their biogenesis, and their role in many cellular phenotypes, it is not fully understood how microRNAs exert their cellular functions because a single microRNA can have hundreds of targets. Hence, identification of microRNA targets is a critical step toward understanding of molecular mechanisms of microRNA-mediated gene expression in normal and disease processes. Here we reported a selection system for microRNA target identification and validation. We used Sleeping Beauty (SB) transposon system to generate selection vectors. The first generation of SB-based vector is named pSSMT-5/6 which carries tetO-EF1a promoter driving GFP/or mCherry-T2A-Pu and tetR-Krab under CMV promoter. We examined the feasibility of these new constructs in 293T and HeLa cells. Using GFP or m-Cherry along with Pu (puromycin resistance) as a reporter, we found that the construct carrying either a given UTR or microRNA sponge provides a significant selection advantage over the vector alone. For example, we cloned 6 different UTRs into the construct and identified 3 of them, CDH1, p53 and p27, revealed stronger mCherry signals and are more resistant to puromycin. Thus, we will test our microRNA library to identify microRNAs capable of targeting these genes (i.e., one known UTR construct against the microRNA library). Together, these results suggest that this system provides proof of principle. In the future, we will generate a UTR library so that we will be able to test for an individual microRNA against the UTR library to identify its biologically relevant targets.

Citation Format: Nanjiang Zhou, Yin-Yuan Mo. A genetic selection system for microRNA targets identification and validation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5286. doi:10.1158/1538-7445.AM2013-5286
Abstract 529: Giα proteins are a differential mediator of activation of RTK signaling in normal and breast cancer cells.
Authors: Wang Zhanwei , Dela-Cruz Rica , Jiang meisheng , Chu Wen-Ming
Published: August 2014

In classic view, inhibitory heterotrimeric G protein alpha subunits 1, 2, and 3 (Gi1α, Gi2α and Gi3α) couple to Giα protein-coupled receptors (GiPCR) and then transduce signals from GiPCRs to downstream pathways in hormone and chemo-attractant signaling. Recent genetic evidence demonstrates that Giα proteins are crucial to embryo development and growth as well as tumorigenesis. However, the molecular mechanism underlying the implication of Giα proteins in this physiological and pathological process is largely unknown.

Here we report that Giα proteins differentially regulate receptor tyrosine kinases (RTKs)-mediated activation of growth factor signaling. Gi1α and Gi3α double deficient (Gi1/3 DKO) mouse embryonic fibroblasts (MEFs), in which Gi2α expression is also lower than that in wild type cells, exhibited a clear defect in ERK1/2 activation by EGF, HB-EGF, insulin and IGF-I, but not aFGF, bFGF or high dose of serum (FBS). Further, Gi1/3 DKO MEFs were largely defective in Akt-mTORC1 activation by aFGF, bFGF, EGF, HB-EGF, serum, but not IGF-1 and insulin. The defect in Akt or ERK1/2 activation in Giα deficient MEFs was rescued upon reintroduction of Gi1α and Gi3α proteins. Interestingly, Giα deficiency impaired activation of Akt-mTROC1 and ERK1/2 by PDGF at a low, but not a high dose. Using a RNA interference strategy, we showed that knockdown of Giα proteins in either wt MEFs or triple negative breast cancer cells (MDA-MB231-D3H2-LN) led to a defect in Akt-mTROC1 and ERK1/2 activation, which is similar to that in Giα deficient cells in response to EGF, bFGF or IGF-1.

Additional experimental analysis revealed that Giα proteins formed a complex with RTKs, Gab1, FRS2 and Shp2 in breast cancer cells in response to EGF, bFGF, or IGF-1. Gi1/3 DKO MEFs showed a defect in EGF- and IGF-1-induced interaction of Gab1 with SHP2, which is required for ERK1/2 activation by EGF and IGF-1, and a defect in bFGF-induced interaction of Grb2 with Gab1, which is required for Akt-mTORC1 activation by aFGF and bFGF. Finally, knockdown of Giα proteins impaired in vitro growth and invasion of breast cancer cells in response to bFGF, EGF, and IGF-1.

Thus, Giα proteins are a differential mediator of RTK signaling in normal and breast cancer cells.

Citation Format: Zhanwei Wang, Rica Dela-Cruz, meisheng Jiang, Wen-Ming Chu. Giα proteins are a differential mediator of activation of RTK signaling in normal and breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 529. doi:10.1158/1538-7445.AM2013-529