bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4738: Clinicopathological roles of adiponectin receptor and leptin receptor in endometrial carcinoma
Authors: Yabushita Hiromitsu , Iwasaki Keita , Ueno Taiki , Wakatsuki Akihiko
Published: May 2015

Objectives:

To clarify the roles of adiponectin receptor (AdipoR) and leptin receptor (ObR) in endometrial carcinoma, expressions of AdipoR-1 and -2 and ObR in endometrial cancer were examined immunohistochemically and relationships to clinicopathological implications were also analyzed.

Methods: Under the written informed consent, paraffin-embedded tissues were obtained from 77 patients with endometrial carcinoma and were stained immunohistochemically using antibodies against AdipoR-1, AdipoR-2 and ObR.

Results: AdipoR-1, AdipoR-2 and ObR were localized predominantly in the cell membrane and cytoplasm of tumor cells and normal endometrial cells. In 77 cases of endometrial cancer, positive expression was observed in 46 cases (59.7%) for AdipoR-1, 47 cases (61.0%) for AdipoR-2 and 33 cases (42.9%) for ObR. Expression of AdipoR-1 was observed more in stage I cases, G1 tumours, tumors with shallow myometrial invasion, tumors negative for lymphovascular space involvement, cases negative for adnexal invasion, and cases with no lymph node metastasis. However, expressions of AdipoR-2 and ObR showed no relationship to any clinicopathological factors. Kaplan-Meier analyses revealed that progression-free and overall survivals were longer in cases with positive AdipoR-1 expression compared with negative AdipoR-1 expression.

Conclusion: Poor expression of AdipoR-1 thus appears to be associated with tumour grade, myometrial invasion, adnexal invasion, lymph-vascular space involvement and lymph-node metastasis, as well as poor prognosis in endometrial cancer.

Citation Format: Hiromitsu Yabushita, Keita Iwasaki, Taiki Ueno, Akihiko Wakatsuki. Clinicopathological roles of adiponectin receptor and leptin receptor in endometrial carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4738. doi:10.1158/1538-7445.AM2014-4738
Abstract 4739: Nutrient depletion caused by PKR induces phosphorylation of AMPK in lung cancer
Authors: Pataer Apar , Guo Chengcheng , Shao Ruping , Correa Arlene M. , Behrens Carmen , Fang Bingliang , Roth Jack A. , Wistuba Ignacio I. , Swisher Stephen G.
Published: May 2015

We have demonstrated that RNA-dependent protein kinase (PKR) and its downstream protein p-eIF2α are independent prognostic markers for survival in lung cancer. In the current study, we aimed to elucidate PKR's specific role in lung cancer. We examined PKR and p-PKR protein expression in 60 frozen primary lung tumor tissues by Western blot analysis and analyzed the association between PKR/p-PKR expression and expression of 139 proteins examined previously by reverse phase protein array in tissue samples from the same 60 patients. We demonstrated several biomarkers that were either positively (p-AMPK) or negatively (IRS1, p-IGF1R, MRE11, ATRIP, telomerase, CHK1, and cyclin E1) correlated with PKR and p-PKR. We confirmed that induction of PKR or p-PKR with expression vectors in lung cancer cells caused phospholation of the AMP-activated protein kinase (AMPK) protein; phospholation was independent of the LKB1, CaMKKβ, and TAK1 pathways. We observed that AMPK phosphorylation was caused by the elevated levels of AMP and decreased levels of ATP resulting from PKR-mediated nutrient depletion. Inhibition of AMPK expression by compound C or siRNA enhanced PKR-mediated cell death. We next investigated the combination of PKR and p-AMPK expression in non-small cell lung cancer patients; we observed that high expression of p-AMPK predicted worse prognosis in patients with high PKR expression but better prognosis in patients with low PKR expression. The p-AMPK may be required for cancer cell survival in patients with high PKR expression and may inhibit cancer cell growth in patients with low PKR expression. In summary, our data indicate that PKR-mediated nutrient depletion induces phosphorylation of AMPK, which is required for lung cancer cell survival.

Citation Format: Apar Pataer, Chengcheng Guo, Ruping Shao, Arlene M. Correa, Carmen Behrens, Bingliang Fang, Jack A. Roth, Ignacio I. Wistuba, Stephen G. Swisher. Nutrient depletion caused by PKR induces phosphorylation of AMPK in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4739. doi:10.1158/1538-7445.AM2014-4739
Abstract 4744: Structure-based development of covalent inhibitors of the activating and T790M gatekeeper mutant forms of the epidermal growth factor receptor (EGFR) leading to the discovery of AZD9291
Authors: Ward Richard A. , Ashton Susan , Anderton Mark , Ballard Pete G. , Bradbury Rob H. , Butterworth Sam , Colclough Nicola , Cross Darren A E , Finlay M Ray V. , McFarland Heather L. , Mellor Martine , Waring Mike J.
Published: May 2015

Small molecule inhibitors of the Epidermal Growth Factor Receptor (EGFR) tyrosine kinase such as gefitinib and erlotinib have been employed successfully in the treatment of non-small cell lung cancer (NSCLC) patients harboring an activating mutation (EGFRm+). However, resistance to these inhibitors in the form of additional mutations such as T790M, (mutation of the gatekeeper residue), is recognized as a clinical issue. This presentation will describe the discovery and evolution of one of our novel chemical series, leading ultimately to the identification of AZD9291, an orally bioavailable, covalent EGFR inhibitor of both the resistance (NCI-H1975, cell phosphorylation IC50 <0.025 uM) and activating mutations (PC9, cell phosphorylation IC50<0.025 uM) that spares inhibition of the wild type form of the receptor (A431, cell phosphorylation IC50>0.5 uM). Wild type EGFR inhibition is believed to drive the observed dose limiting toxicities (such as skin rash and diarrhea) for these first generation therapies in the clinic. New data will be presented for the first time including a broader description of the medicinal chemistry program that led to the identification of AZD9291. We shall also present previously undisclosed work on the identification of additional distinct chemical series and an update of recent data from ongoing AZD9291 Phase I clinical studies in NSCLC patients.

L858R/T790M Double Mutant (resistance) cell IC50 uMEGFRm+ Single Mutant (activating) cell IC50 uMEGFR Wild Type cell IC50 uMAqueous solubility, (salt) (pH=6.8, ug/mL)L858R/T790M Double Mutant efficacy (% TGI at 5mpk PO QD for 14 days)<0.025<0.025>0.5>490119

Citation Format: Richard A. Ward, Susan Ashton, Mark Anderton, Pete G. Ballard, Rob H. Bradbury, Sam Butterworth, Nicola Colclough, Darren A E Cross, M Ray V. Finlay, Heather L. McFarland, Martine Mellor, Mike J. Waring. Structure-based development of covalent inhibitors of the activating and T790M gatekeeper mutant forms of the epidermal growth factor receptor (EGFR) leading to the discovery of AZD9291. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4744. doi:10.1158/1538-7445.AM2014-4744
Abstract 475: GATA3 mutations in breast cancer
Authors: Takaku Motoki , Adomas Aleksandra , Grimm Sara A. , Takashi Shimbo , Wade Paul A.
Published: May 2015

Breast cancer is one of the most common cancers in women worldwide, resulting in over 400,000 deaths per year. Approximately 90% of breast cancers occur in women who have no family history of breast cancer, indicating that most breast cancers occur following spontaneous mutations resulting from the aging process or the action of environmental agents. Breast cancers can be divided into several subtypes base on gene expression profiles. Luminal breast cancer, the most common subtype, is characterized by expression of estrogen receptor alpha (ER-alpha). The zinc finger transcription factor GATA3, which is known as a regulator of mammary gland development, is frequently expressed in luminal breast cancer cells, and its expression is highly correlated with ER-alpha. In breast cancers, the expression levels of GATA3 directly correlate with favorable prognosis, and its ectopic expression leads to the suppression of tumor metastasis. Comprehensive genomic analysis of breast tumors has revealed frequent somatic mutations of GATA3 in luminal breast cancers. However it is unclear how these GATA3 mutations affect the biological properties of breast cancer. These mutations are located at exclusively in exons 5 and 6 that encode the carboxyl terminus of the protein. Exon 5 contains the second of two zinc finger domains (ZnF2), which is important for the DNA binding activity of GATA3. Exon 6 has no previously described biochemical function.

In this study, we focus on two mutations found in cancer patients - a frame shift at tyrosine 345 (Y345fs) and a frame shift at arginine 330 (R330fs). These two mutants are of particular interest as they produce truncated proteins, and both mutants lack exon 6. Y345fs preserves ZnF2, while R330fs interrupts ZnF2. These two mutants permit dissection of the molecular outcomes flowing from loss of exon 6 (Y345fs) versus the combination of loss of ZnF2 and exon 6 (R330fs). To assess the impact of these mutations, we established breast cancer cells expressing Y345fs and R330fs respectively, and analyzed their phenotype at the cellular and molecular levels. These studies begin to address the biochemical and functional outcomes of commonly occurring mutations in breast cancer.

Citation Format: Motoki Takaku, Aleksandra Adomas, Sara A. Grimm, Shimbo Takashi, Paul A. Wade. GATA3 mutations in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 475. doi:10.1158/1538-7445.AM2014-475
Abstract 4757: A novel class of selective estrogen receptors degraders regresses tumors in pre-clinical models of endocrine-resistant breast cancer
Authors: Joseph James D. , Darimont Beatrice , Govek Steven , Brigham Dan , Qian Jing , Sensintaffar John , Shao Gang , Aparicio Anna , Kahraman Mehmet , Lai Andiliy , Lee Kyoung-Jin , Lu Nhin , Nagasawa Johnny , Moon Michael , Rix Peter , Smith Nick , Hager Jeff
Published: May 2015

80% of all breast cancers express the estrogen receptor alpha (ERα) and thus are treated with anti-hormonal therapies that directly block ER function (e.g.Tamoxifen) or hormone synthesis (Aromatase Inhibitors). While these therapies are initially effective, acquired resistance invariably emerges and disease progression ensues. Importantly, the majority of these tumors continue to depend on ERα for growth and survival via both ligand-dependent and ligand-independent pathways. The emerging evidence that ERα can be activated in the absence of estrogens via point mutations in ERα or cellular signaling pathways supports the development of agents that are not only competitive ERα antagonists but also reduce steady state levels of the receptor and thus limit both ligand dependent and independent signaling.

We have identified two novel series of non-steroidal ERα antagonists, series I exemplified by ARN-810, now in clinical trials for treatment of endocrine resistant breast cancer, and series II, both of which induce degradation of ERα at picomolar concentrations resulting in significant reduction in steady state ERα protein levels in breast cancer cell lines. Using peptide-based conformational profiling, we show that both series induce ERα conformations that are distinct from both fulvestrant and tamoxifen indicating novel mechanism of action. In vitro, both ligand series are active on wild-type and the constitutively active ERα mutants found in endocrine resistant breast cancer patients. Importantly, these compounds yield tumor regression in both tamoxifen-sensitive and -resistant models of breast cancer in vivo. Based on their unique in vitro profile, and good pharmacokinetics following oral dosing, these compounds represent a novel class of Selective Estrogen Receptor Degraders (SERDs) that hold promise as a next generation therapy for the treatment of ER+ breast cancer as monotherapy, as well as in combination with agents that target other pathways involved in both intrinsic and acquired endocrine resistance.

Citation Format: James D. Joseph, Beatrice Darimont, Steven Govek, Dan Brigham, Jing Qian, John Sensintaffar, Gang Shao, Anna Aparicio, Mehmet Kahraman, Andiliy Lai, Kyoung-Jin Lee, Nhin Lu, Johnny Nagasawa, Michael Moon, Peter Rix, Nick Smith, Jeff Hager. A novel class of selective estrogen receptors degraders regresses tumors in pre-clinical models of endocrine-resistant breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4757. doi:10.1158/1538-7445.AM2014-4757
Abstract 477: The dynamic changes of KLF4 in the oral squamous cell carcinogenesis
Authors: Liu Man , Su Ying , Zhang Xinyan
Published: May 2015

KLF4 (formerly GKLF) is a zinc-finger transcription factor expressed in the epithelia of the skin, lungs, gastrointestinal tract and several other organs. In vitro studies have suggested that KLF4 plays an important role in cell proliferation and/or differentiation. Several studies implied that KLF4 is associated with both tumour suppression and oncogenesis, but the function of KLF4 in the oral carcinogenesis has not been investigated. To address this issue we have generated an animal model of experimental tongue cancerous lesions in C57BL/6J mouse by using carcinogen 4-nitroquinoline-1-oxide (4NQO) in drinking water .And we detected the KLF4 expression in mouse tongue precancerous and cancer lesions by immunohistochemistry. Normally KLF4 was strongly expressed in the nuclei of mice tongue epithelial cells. The staining of KLF4 was gradually decreased from normal epithelial to hyperplasia to dysplasia, and markedly decreased in the squamous cell carcinomas. The results indicated that KLF4 could served as a tumour-suppressor functions in the oral carcinogenesis.

Citation Format: Man Liu, Ying Su, Xinyan Zhang. The dynamic changes of KLF4 in the oral squamous cell carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 477. doi:10.1158/1538-7445.AM2014-477
Abstract 4772: Novel transforming ErbB2 mutations in leukemia
Authors: Keck Jamie , Druker Brian
Published: May 2015

The ErbB2/Her2 type 1 transmembrane receptor tyrosine kinase heterodimerizes with ligand-bound EGFR, ErbB3, or ErbB4 to activate multiple signaling pathways. ErbB2 is commonly amplified in breast, stomach, cervical, and bladder tumors and point mutations are found in many cancers. However, little is known about aberrant ErbB2 in hematologic malignancies. Deep sequencing was performed on primary leukemia patient samples and point mutations were identified within the extracellular receptor binding domains and the intracellular C-terminal region of ErbB2. Expression of these mutations in NIH 3T3 fibroblasts exhibited a transformed morphological phenotype and in Ba/F3 pro-B cells led to IL-3-independent growth and differential signaling within the MAPK and PI3K pathways. Further characterization of the mutations will assess migration properties, differential sensitivity to ErbB-family drug inhibitors, and heterodimer formation using bimolecular fluorescence complementation microscopy. Overall, this work identifies novel transforming ErbB2 mutations and may provide insight into effective therapy for leukemia patients harboring ErbB2 mutations.

Citation Format: Jamie Keck, Brian Druker. Novel transforming ErbB2 mutations in leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4772. doi:10.1158/1538-7445.AM2014-4772
Abstract 4773: Inhibition of mutant EGFR in lung cancer cells triggers SOX2-FOXO6 dependent survival pathways
Authors: Rothenberg Stephen Michael , Concannon Kyle , Cullen Sarah , Turke Alexa B. , Faber Anthony C. , Engelman Jeffrey A. , Maheswaran Shyamala , Haber Daniel A.
Published: May 2015

Treatment of EGFR-mutant lung cancer with erlotinib results in dramatic tumor regression but it is invariably followed by drug resistance. In characterizing early transcriptional changes following drug treatment of mutant EGFR-addicted cells, we identified the stem cell transcriptional regulator SOX2 as being rapidly and specifically induced, both in vitro and in vivo. Suppression of SOX2 sensitizes cells to erlotinib-mediated apoptosis, whereas its ectopic expression reduces drug-induced cell death. We show that erlotinib relieves EGFR-dependent suppression of FOXO6, leading to SOX2 induction and repression of the pro-apoptotic BH3-only genes BIM and BMF. Together, these observations point to a physiological feedback mechanism that attenuates oncogene addiction-mediated cell death and may contribute to the acquisition of drug resistance.

Citation Format: Stephen Michael Rothenberg, Kyle Concannon, Sarah Cullen, Alexa B. Turke, Anthony C. Faber, Jeffrey A. Engelman, Shyamala Maheswaran, Daniel A. Haber. Inhibition of mutant EGFR in lung cancer cells triggers SOX2-FOXO6 dependent survival pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4773. doi:10.1158/1538-7445.AM2014-4773
Abstract 4774: The antitumor effects of PI3K beta inhibitors in PTEN negative prostate cancer are enhanced by inhibition of reactivated PI3K alpha signaling
Authors: Schwartz Sarit , Carver Brett S. , Wongvipat John , Rodrik-Outmezguine Vanessa , Stanchina Elisa D. , Trigwell Cath , Barry Simon , Baselga Jose , Chandarlapaty Sarat , Scher Howard I. , Sawyers Charles L. , Rosen Neal
Published: May 2015

The PI3K pathway is dysregulated in many cancers via selective activation of class 1 isoforms. In tumors with deficient PTEN function, signaling is driven by PI3K beta. We show here that a selective inhibitor of PI3K beta inhibits the AKT/mTOR pathway in tumors with defective PTEN function, but is ineffective in those where the pathway is driven by receptor tyrosine kinases. However, inhibition of PI3K signaling by PI3K beta inhibitors is limited by relief of AKT/mTOR dependent feedback and reactivation of IGF1R and other receptors. This results in activation of PI3K alpha and a rebound of PI3K-AKT signaling. This rebound is suppressed and tumor cell inhibition is enhanced with combined inhibition of PI3K alpha and beta. Combined administration of isoform selective PI3K inhibitors may more effectively inhibit the pathway than pan-PI3K inhibitors because of the greater selectivity and decreased off-target toxicity of the former.

In PTEN deficient models of prostate cancer, triple therapy with PI3K alpha and beta selective inhibitors combined with a potent androgen receptor inhibitor suppresses the reciprocal feedback activation of both pathways and results in marked (complete) eradication of tumors in vivo.

Citation Format: Sarit Schwartz, Brett S. Carver, John Wongvipat, Vanessa Rodrik-Outmezguine, Elisa De Stanchina, Cath Trigwell, Simon Barry, Jose Baselga, Sarat Chandarlapaty, Howard I. Scher, Charles L. Sawyers, Neal Rosen. The antitumor effects of PI3K beta inhibitors in PTEN negative prostate cancer are enhanced by inhibition of reactivated PI3K alpha signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4774. doi:10.1158/1538-7445.AM2014-4774
Abstract 478: Predicting interactome networks perturbations in human cancer: application to gene fusions in acute lymphoblastic leukemia
Authors: Hajingabo Leon , Daakour Sarah , Martin Maud , Grausenburger Reinhard , Panzer-Grümayer Renate , Dequiedt Franck , Helden Jacques Van , Twizere Jean-Claude , Simonis Nicolas
Published: May 2015

Genomic variations such point mutations and gene fusions are directly or indirectly associated with human diseases. They are recognized as diagnostic, prognostic markers and as therapeutic targets. However, predicting the functional impact of these genetic alterations beyond affected genes and their products is challenging because diseased phenotypes likely result in complex molecular interaction networks. Using as models three different chromosomal translocations ETV6-RUNX1 (TEL-AML1), BCR-ABL1, and E2A-PBX1 (TCF3-PBX1) frequently found in B-cell acute lymphoblastic leukemia (B-ALL), we develop an approach to extract from gene expression changes, perturbed molecular interactions. We show that the Myc and JunD transcriptional circuits are specifically deregulated following TEL-AML1 and TCF3-PBX1 gene fusions, respectively. We also identified the bulk mRNA NXF1-dependent machinery as a direct target for the TCF3-PBX1 fusion protein. Through a novel approach combining gene expression and interactome data analysis, we provide new insight into TCF3-PBX1 and TEL-AML1 acute lymphoblastic leukemia.

Note: This abstract was not presented at the meeting.

Citation Format: Leon Hajingabo, Sarah Daakour, Maud Martin, Reinhard Grausenburger, Renate Panzer-Grümayer, Franck Dequiedt, Jacques Van Helden, Jean-Claude Twizere, Nicolas Simonis. Predicting interactome networks perturbations in human cancer: application to gene fusions in acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 478. doi:10.1158/1538-7445.AM2014-478
Abstract 4784: Selective bioluminogenic HDAC activity assays for profiling HDAC inhibitors
Authors: Kupcho Kevin R. , Evans Nathan J. , Niles Andrew L. , Kirkland Thomas A. , Lazar Dan F.
Published: May 2015

Histone deacetylases (HDACs) play critical roles in the regulation of gene transcription and cell signaling events by deacetylating histones and other important non-histone substrates. Aberrant increases in HDAC enzyme activities are therefore implicated in a number of human infirmities, including cancers, metabolic disease and neurodegeneration. Fortunately, HDAC enzymes represent attractive pharmacological targets because they are readily tractable with small molecule inhibitors. In fact, several HDAC inhibitors (HDACi) have recently proceeded through (or are near) the FDA approval process for the treatment of hematologic malignancies. However, the promise of clinical HDACi therapy has been hampered by significant dose-limiting toxicities. These off-target effects have led to a renewed focus on basic HDAC biology and the development of isoenzyme-specific HDAC inhibitors which could avoid off-target effects. To help facilitate the discovery of compounds with better defined selectivity profiles, we have developed lysine deacetylase assays that selectively measure specific isoenzyme activities in cells, extracts, or purified recombinant preparations. These assays are based on substrates that are selective due to a combination of extended peptide sequence and novel chemical modifications. Deacetylase activity is measured by delivering a single, pro-luminogenic, homogeneous assay reagent to assay wells, resulting in luminescence proportional to HDAC activity. In addition to being isoenzyme selective, these novel substrates are cell permeable allowing for lytic and non-lytic cell-based HDAC assays. Lastly, these assays are also fully compatible with fluorescent viability and/or cytotoxicity assays. This provides additional flexibility for multiplexed formats which examine not only selective HDAC inhibition, but the functional consequences they exert on cell health.

Citation Format: Kevin R. Kupcho, Nathan J. Evans, Andrew L. Niles, Thomas A. Kirkland, Dan F. Lazar. Selective bioluminogenic HDAC activity assays for profiling HDAC inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4784. doi:10.1158/1538-7445.AM2014-4784
Abstract 4785: miR-125b targets erythropoietin and its receptor and their expression correlates with metastatic potential and ERBB2/HER2 expression
Authors: Ferracin Manuela , Bassi Cristian , Pedriali Massimo , Pagotto Sara , D'Abundo Lucilla , Zagatti Barbara , Volpato Stefano , Querzoli Patrizia , Negrini Massimo
Published: May 2015

Background: The microRNA 125b is a double-faced gene expression regulator described both as a tumor suppressor gene (in solid tumors) and an oncogene (in hematologic malignancies). In human breast cancer, it is one of the most down-regulated miRNAs and is able to modulate ERBB2/3 expression. Here, we investigated its targets in breast cancer cell lines after miRNA-mimic transfection. We examined the interactions of the validated targets with ERBB2 oncogene and the correlation of miR-125b expression with clinical variables.

Methods: MiR-125b possible targets were identified after transfecting a miRNA-mimic in MCF7 cell line and analyzing gene expression modifications with Agilent microarrays and Sylamer bioinformatic tool. Erythropoietin (EPO) and its receptor (EPOR) were validated as targets of miR-125b by luciferase assay and their expression was assessed by RT-qPCR in 42 breast cancers and 13 normal samples. The molecular talk between EPOR and ERBB2 transcripts, through miR-125b, was explored transfecting MDA-MD-453 and MDA-MB-157 with ERBB2 RNA and using RT-qPCR.

Results: We identified a panel of genes down-regulated after miR-125b transfection and putative targets of miR-125b. Among them, we validated erythropoietin (EPO) and its receptor (EPOR) - frequently overexpressed in breast cancer - as true targets of miR-125b. Moreover, we explored possible correlations with clinical variables and we found a down-regulation of miR-125b in metastatic breast cancers and a significant positive correlation between EPOR and ERBB2/HER2 levels, that are both targets of miR-125b and function as competing endogenous RNAs (ceRNAs).

Conclusions: Taken together our results show a mechanism for EPO/EPOR and ERBB2 co-regulation in breast cancer and confirm the importance of miR-125b in controlling clinically-relevant cancer features.

Citation Format: Manuela Ferracin, Cristian Bassi, Massimo Pedriali, Sara Pagotto, Lucilla D'Abundo, Barbara Zagatti, Stefano Volpato, Patrizia Querzoli, Massimo Negrini. miR-125b targets erythropoietin and its receptor and their expression correlates with metastatic potential and ERBB2/HER2 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4785. doi:10.1158/1538-7445.AM2014-4785
Abstract 4786: Circulating miR-21, miR-146a and miR-210 levels in plasma are associated with clinical outcome in breast cancer
Authors: Sourvinou Ioanna S. , Markou Athina , Malamos Nikolaos , Georgoulias Vasilis , Lianidou Εvi S.
Published: May 2015

Introduction: The discovery of novel non-invasive tumor biomarkers for early diagnosis, prognosis and therapy monitoring remains an undeniable imperative necessity. Circulating miRNAs have recently attracted a lot of interest as novel promising tumor biomarkers. In the present study, we investigated the expression levels of three cancer-associated miRNAs in plasma and evaluated their prognostic significance in breast cancer.

Materials and Methods: We examined the expression levels of miR-21, miR-146a and miR-210 in plasma samples of 134 breast cancer patients (57 patients with verified metastasis and 77 patients with operable breast cancer) and 20 healthy individuals. Circulating miRNAs were isolated from 200μL of plasma using mirVana PARIS kit and in each sample 25fmol of the synthetic cel-miR-39 were spiked as exogenous control. For the quantification of plasma miRNAs, a stem-loop cDNA approach and TaqMan real time PCR (LightCycler 2, Roche Applied Science) were used. The normalization procedure was based on a combination of cel-miR-39 as exogenous control and hsa-miR-191 as endogenous control. Relative expression of circulating miRNAs in plasma was evaluated as previously described (Sourvinou et al., J. of Mol. Diagn, 2013).

Results: Our results indicate that the expression levels of all three circulating miRNAs are significantly increased in plasma both in operable and metastasis verified breast cancer patients in comparison to those of healthy individuals (p<0.001). The expression levels of plasma miR-210 are slightly increased in patients with operable breast cancer in respect to patients with verified metastasis (p=0.034). In patients with verified metastasis, Kaplan-Meier survival analysis demonstrated an association between elevated levels of plasma miR-21 and overall survival (OS) (p= 0.001) and there was a trend with disease-free interval (DFI) (p=0.053). Concerning miR-146a, increased plasma expression levels were correlated with short OS (p=0.029), but there was no correlation with DFI (p=0.914). Finally, respecting miR-210, Kaplan-Meier survival analysis did not show any association with OS or DFI of these patients.

Conclusions: Our results indicate that quantification of circulating miR-21 and miR-146a in plasma are promising prognostic biomarkers in breast cancer and should be further evaluated in a large number of patients.

Citation Format: Ioanna S. Sourvinou, Athina Markou, Nikolaos Malamos, Vasilis Georgoulias, Εvi S. Lianidou. Circulating miR-21, miR-146a and miR-210 levels in plasma are associated with clinical outcome in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4786. doi:10.1158/1538-7445.AM2014-4786
Abstract 4791: The role of microRNA-15a/16 in early B1 cell development in a mouse model of chronic lymphocytic leukemia
Authors: Underbayev Chingiz , Kasar Siddha , Degheidy Heba , Marti Gerald , Lightfoote Marilyn , Raveche Elizabeth
Published: May 2015

New Zealand Black (NZB) mouse is a de-novo model of chronic lymphocytic leukemia (CLL) that has been studied as a model of B-cell lymphoproliferative disorder. In most human CLL and NZB there is decreased expression of the microRNA miR15a/16. In NZB (and some CLL patients) this is due a point mutation six bases downstream from pre-miR-16 region. In both NZB mouse model and human CLL, the disease is characterized by the presence of a malignant clone of B-1 cells expressing dull levels of CD5 (T cell marker), CD11b (myeloid marker) and B220 (B cell marker). In this report we investigated the molecular pathways that result in increased B-1 lineage development in the mouse model of CLL. Initially we observed that in NZB, the B-1 lineage cells had reduced expression of Pu.1 when compared to NZB B-2 cells. PU.1 is a transcription factor required for B lineage differentiation and PU.1 knockout mice were previously found by others to have only B-1 lineage cell differentiation. To study early B1/B2 divergent cell development pathways in the context of CLL and shed light on the potential malignant cell origin of this disease, we generated induced pluripotent stem cells (iPS) from NZB spleen stromal fibroblasts. Our in vivo and in vitro studies on NZB iPS and hematopoietic progenitors (LSK) differentiation towards B-cell lineage cells followed by multicolor flow based cell sorting revealed a substantial block in the maturation of NZB iPS cells compared to wild type counterparts. Preliminary data using congenic NZB mice with corrected mir-15a/16-1 locus demonstrated that normal levels of miR-15a/16 regulate B-cell differentiation towards a higher expression of PU.1 and B220 (CD45R) and lower levels of CD11b suggestive of an enhanced B2 cell maturation pathway. Our results support the hypothesis that decreased miR-15a plays a role in B1 lymphocyte development bias in the course of B cell maturation by regulating the expression of a number of genes such as B220, PU.1 and CD11b. This work will help further uncover early molecular events which determine the increased B1 versus B2 lineage commitment associated with low levels of miR-15a/16 as an initial lesion in the development of CLL.

Citation Format: Chingiz Underbayev, Siddha Kasar, Heba Degheidy, Gerald Marti, Marilyn Lightfoote, Elizabeth Raveche. The role of microRNA-15a/16 in early B1 cell development in a mouse model of chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4791. doi:10.1158/1538-7445.AM2014-4791
Abstract 4794: NANOG represses mitochondrial energy production and promotes fatty acid synthesis to promote self-renewal in tumor-initiating cells
Authors: Chen Chia-Lin , Punj Vasu , Sher Linda , Petrovic Lydia , Machida Keigo
Published: May 2015

Stem cell markers such as Nanog have been implicated in various cancer, but whether they are functionally contributing to cancer pathogenesis has remained unclear. We previously reported that Toll-like receptor 4 (TLR4) signaling regulated NANOG expression in liver cancers (Chen et al., J. Clin. Inv., 2013). Here, we determined novel targets of NANOG in CD133+CD49f+ tumor-initiating cells (TICs) from patient and mouse models of hepatocellular carcinoma (HCC) using genome-wide NANOG-binding site analysis (ChIP-seq). Silencing NANOG inhibits tumor development in HCC mouse models and genesis of TICs as alcohol/obesity-associated increase of serum bacterial products activates TLR4 signaling. To determine novel targets of NANOG in TICs isolated from several HCC models, ChIP-seq in TICs was performed. NANOG binds genes of oxidative phosphorylation and β-oxidation in mitochondria. Silencing NANOG promotes oxidative phosphorylation and β-oxidation, indicating that NANOG is a suppressor of mitochondria-mediated energy production. We identified the TLR4-NANOG oncogenic pathway in the genesis of TICs and liver tumor in HCC mouse models, including alcohol/high-fat-diet-fed hepatitis C virus (HCV) transgenic mice, DEN/phenobarbtal-treated and Spnb2+/- mice. We identified the stimulation of TLR4 transactivates Nanog through E2F1 phosphorylation at serine residues 332/336 and generates TICs, leading to development of liver tumors. Metabolomic analysis demonstrates metabolic reprogramming in TICs through NANOG. Reduction of NANOG expression resulted in an improved cellular energy balance, including reduction of the glycolysis pathway, increased fatty acid consumption and greater uptake of amino acids. The improved energy status is linked to a shift away from glycolysis and a greater reliance on fatty acid β-oxidation and amino acid anaplerosis. Taken together, these results suggest that NANOG-mediated metabolic reprogramming through suppression of mitochondria function in both experimental and clinical HCC downstream of TLR4/NANOG generates TICs and drives liver tumorigenesis.

Citation Format: Chia-Lin Chen, Vasu Punj, Linda Sher, Lydia Petrovic, Keigo Machida. NANOG represses mitochondrial energy production and promotes fatty acid synthesis to promote self-renewal in tumor-initiating cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4794. doi:10.1158/1538-7445.AM2014-4794
Abstract 4795: CCK2R marks gastric antral stem cell and mediates antral carcinogenesis
Authors: Yoku Hayakawa , Jin Guanchun , Wang Hongshan , Chen Xiaowei , Westphalen Christoph B. , Asfaha Samuel , Worthley Daniel L. , Renz Bernhard , Ariyama Hiroshi , Dubeykovskaya Zinaida A. , Takemoto Yoshihiro , Mulay Ashlesha , Tailor Yagnesh , Chen Duan , Muthupalani Sureshkumar , Fox James G. , Takaishi Shigeo , Wang Timothy C.
Published: May 2015

Gastrin is a hormone that binds to the CCK2 receptor and promotes proximal gastric cancer, but inhibits distal gastric cancer development. However, the precise roles of the CCK2 receptor, and its alternative ligand, progastrin, in gastric carcinogenesis have not been clarified. In this study, we found that progastrin accelerated antral proliferation and carcinogenesis through CCK2R+ antral stem cell expansion, using mouse gastric cancer models and transgenic mice lines including human progastrin-overexpressing (hGAS) mice, CCK2R knockout mice, Lgr5-CreERT-IRES-GFP knockin mice, and newly generated CCK2R-BAC-CreERT mice. Progastrin promoted gastric antral cancer development induced by MNU and/or H. felis. During carcinogenesis, progastrin increased the expression of Lgr5 and gland fission in response to the chemical carcinogen MNU. Genetic ablation of CCK2R diminished these progastrin-mediated effects. In vitro 3D culture experiments revealed that progastrin, but not amidated gastrin, significantly increased gastric organoid formation and growth in Noggin-free condition, effects that were ablated by a CCK2R inhibitor YF-476 or CCK2R gene deletion. In the antrum, CCK2R was expressed in an Lgr5low cell population that displayed stemness, which could be enhanced by progastrin. In the presence of progastrin, Lgr5lowCCK2R+ cells interconverted to Lgr5hi cells. Finally, we generated a new BAC-transgenic CCK2R-CreERT murine line, and lineage tracing experiments showed that CCK2R+ cells, which reside slightly above the base of the antrum, contained long-lived stem cells in vivo and in vitro. Chemical inhibition of CCK2R attenuated progastrin-dependent cancer development in mice. In conclusion, CCK2R labels Lgr5low antral stem cells that can be activated and expanded by progastrin. These findings may help the understanding of the underlying mechanism in gastric stem cell regulation by a CCK2R signal.

Citation Format: Hayakawa Yoku, Guanchun Jin, Hongshan Wang, Xiaowei Chen, Christoph B. Westphalen, Samuel Asfaha, Daniel L. Worthley, Bernhard Renz, Hiroshi Ariyama, Zinaida A. Dubeykovskaya, Yoshihiro Takemoto, Ashlesha Mulay, Yagnesh Tailor, Duan Chen, Sureshkumar Muthupalani, James G. Fox, Shigeo Takaishi, Timothy C. Wang. CCK2R marks gastric antral stem cell and mediates antral carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4795. doi:10.1158/1538-7445.AM2014-4795
Abstract 4803: Cancer-associated fibroblast promotes gastric cancer invasion and epithelial-mesenchymal transition via the IL-6/JAK/STAT3 signaling pathway
Authors: Su Liping , Wu Xiongyan , Zhou Quan , Wang Chenchen , Liu Bingya
Published: May 2015

Objectives: The molecular mechanisms involved in the regulation of gastric cancer progression by CAFs are still unclear. The aim of the present study was to determine if CAFs can enhance the tumor epithelial-to-mesenchymal transition (EMT) and invasion of gastric cancer cells, and link CAFs with activation of the IL-6/JAK/ STAT3 signaling pathway in the progression of gastric cancer.

Methods: Fibroblasts were isolated from gastric cancer. The identification of CAFs was defined by immunofluorescence staining and qRT-PCR. IL-6 expression in CAFs and gastric cancer cells was detected by qRT-PCR and ELISA. EMT and activation of Stat3 pathway in gastric cancer cells were detected by realtime PCR and western blot. Cell invasion assays were performed by using 8 μm transwell chambers.

Results: IL-6 expression in gastric cancer tissue was significantly higher than that in adjacent non-tumor tissue (3.450ng/g vs 2.665ng/g,P<0.01). Using real time RT-PCR and ELISA, we found IL-6 expression was significantly higher in CAFs compared to that in gastric cancer cells. Co-culture with CAFs in a transwell system induced EMT of gastric cancer cells, which was demonstrated by decreased expression of E-cadherin and α-catenin, and increased expression of Twist1, MMP2 and ZEB2. However, deprivation of IL-6 using a neutralizing antibody or inhibition Stat3 activation with AG490 impaired the EMT of gastric cancer cells. Gastric cancer cells co-cultured with CAFs showed enhanced ability of migration and invasion than gastric cancer cells alone. However, adding neutralizing IL-6 antibody or AG490 into the co-culture system led to significantly decreased invasion (P < 0.05) of SGC-7901 cells. Moreover, IL-6 secreted from CAFs activated the JAK/STAT3 pathway in gastric cancer cells, however, EMT and invasion of gastric cancer cells was inhibited when IL-6/JAK/STAT3 pathway was blocked.

Conclusions: CAFs induced EMT and invasion of gastric cancer cells through activation of the IL-6/JAK/STAT3 pathway.

Citation Format: Liping Su, Xiongyan Wu, Quan Zhou, Chenchen Wang, Bingya Liu. Cancer-associated fibroblast promotes gastric cancer invasion and epithelial-mesenchymal transition via the IL-6/JAK/STAT3 signaling pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4803. doi:10.1158/1538-7445.AM2014-4803
Abstract 4817: Circulating tumor cells enriched by the negative selection system PowerMag is a useful predictor for treatment response of metastatic colorectal cancer patients
Authors: Hsieh Jason Chia-Hsun , Lin Hung-Chih , Hsu Hung-Chih , Chang John Wen-Cheng , Tseng Ching-Ping
Published: May 2015

Enumeration of circulating tumor cells (CTCs) provides prognostic and predictive value to guide treatment options. This study aims to (1) evaluate the merits of the PowerMag system, an immunomagnetic separation technique that we recently developed for negative selection of CTCs, in the prediction of treatment response; (2) perform analysis to investigate whether there is a correlation between the imaging response and the changes of CTCs counts before and during chemotherapy for the patients with metastatic colorectal cancer (mCRC). A total of 47 mCRC patients were enrolled in this prospective single-center study. CTCs were isolated from the peripheral blood of these patients by PowerMag at baseline and 2 weeks after starting first dose of chemotherapy. CTCs enumeration was then performed by immunofluorescence staining using the anti-epithelial cell adhesion molecule (EpCAM) antibody. Tumor response at 12 weeks after chemotherapy of the whole cohort was monitored by computed tomography (CT) scans following RECIST criteria 1.0. Our data revealed that PowerMag is a cost-effective negative selection-based CTCs isolation platform with a CTCs detection rate of 89.4% (42/47). The CTCs counts could successfully differ mCRC patients from healthy donors (P = 0.0015). The higher baseline CTCs number was associated with initially metastatic status rather than recurrent settings (P = 0.042). In all assessable patients, a high correlation rate of 86.7% was found between changes of CTCs (decline or elevation) and CT responses (progressive disease, and partial response/stable disease) with a P value of 0.005 (Fisher exact test). These data indicate that the change of CTCs counts before and during treatment is in accord with the image response evaluation at 12 weeks after initiation of treatment. In conclusion, PowerMag is a cost-effective method for negative selection of CTCs from cancer patients. Moreover, comparison of CTCs counts before and during treatment is a useful predictor for disease progression of mCRC patients that warrants further investigation in large-scale validation trial.

Citation Format: Jason Chia-Hsun Hsieh, Hung-Chih Lin, Hung-Chih Hsu, John Wen-Cheng Chang, Ching-Ping Tseng. Circulating tumor cells enriched by the negative selection system PowerMag is a useful predictor for treatment response of metastatic colorectal cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4817. doi:10.1158/1538-7445.AM2014-4817
Abstract 4821: Circulating tumor cells: A key deliverable in oncology clinical trials
Authors: Xue Chengsen , Cortes Kasia E. , Hura Dorina , Baptiste Melissa L. , Renato Paradiso , Cuomo Joanne , Closkey Thomas W. Mc
Published: May 2015

Circulating tumor cell (CTC) enumeration is a new pharmacodynamic biomarker developed from an emerging rare event technology showing prognostic potential for the management and monitoring of patients with colon, breast, and prostate cancer. As more evidence is gradually accumulating at multiple cancer institutes, CTC has the potential to become a surrogate marker for cancer prognosis. There are challenges in performing CTC testing, however this assay has the opportunity to become a key deliverable in oncology clinical trials.

Our experience in the implementation of CTC testing in clinical trials over the past four years has led to recommendations for future CTC applications, including critical aspects such as sample accessioning and logistics, operational maintenance and method training, data collection and sufficient IT infrastructure to accommodate large data files in full scale trial operations. To have a successful trial using the CTC test as a monitored biomarker, the estimation of sample size for the clinical trial has to take into consideration the anticipated disqualification rate of collected samples. In our clinical trial experiences, 10% of samples submitted were disqualified for reasons ranging from insufficient sample volume to severe hemolysis. Sample logistics and transportation considerations are keys to successful sample management. Strict quality control (QC) measurements, a robust training program, effective data management, and proper maintenance of the CTC system are keys for delivering standardized results in a timely manner.

Our results indicate that CTC testing can be effectively incorporated into large scale clinical trials and the data can be seamlessly integrated into the Laboratory Information Management System (LIMS). CTC results may offer clinically useful information about patients and their response to therapy. From a patient perspective, the CTC test only requires a tube of blood and is well suited to multiple sampling time points during a clinical trial.

Citation Format: Chengsen Xue, Kasia E. Cortes, Dorina Hura, Melissa L. Baptiste, Paradiso Renato, Joanne Cuomo, Thomas W. Mc Closkey. Circulating tumor cells: A key deliverable in oncology clinical trials. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4821. doi:10.1158/1538-7445.AM2014-4821