bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3583: Reducing GC-bias and improving coverage distribution in Illumina sequencing using the Kapa Biosystems library construction method
Authors: Thorner Aaron R. , Sunkavalli Ashwini , Lin Ling , Jones Robert T. , Schubert Laura , Ducar Matthew D. , Adusumilli Ravali , Dolcen Deniz Nesli , Ziaugra Liuda , Lepine Jack , MacConaill Laura E. , Hahn William C. , Meyerson Matthew , Hummelen Paul Van
Published: May 2015

Next-generation sequencing (NGS) is increasingly used in the laboratory and clinic to identify genomic factors contributing to tumorigenesis, such as somatic mutations, DNA insertions and deletions, transcriptome and epigenetic changes, and chromosomal abnormalities. Because tumor specimen tissues are often quite limited or are from rare and precious samples, it is necessary to prepare sequencing libraries from minimal amounts of DNA. Consequently, limited DNA input necessitates amplification during NGS library preparation. Therefore, it is of great importance to amplify the library in an efficient and uniform manner to achieve reproducible amplification of the library fragments and limit GC content bias. Variable GC-bias among libraries is of particular importance for copy number variant (CNV) detection based on sequence coverage.

Here, we compare the TruSeq DNA Sample Preparation Kit (Illumina Inc., San Diego, CA) and the KAPA Library Preparation Kit (Kapa Biosystems, Wilmington, MA) to determine which technique achieves optimal library coverage. The major difference between the methods is the DNA polymerase used for library enrichment. The Kapa HiFi polymerase was engineered to have higher fidelity and increased DNA affinity relative to the Herculase II Fusion DNA Polymerase (Agilent Technologies, Inc., Santa Clara, CA) that we used for library enrichment with the TruSeq kit. Thus, the Kapa HiFi polymerase should more efficiently amplify targets across a larger GC content range and improve sequence coverage uniformity.

Relative to TruSeq-prepared libraries, the Kapa-prepared libraries had a larger mean insert size, more uniform coverage distribution, and better coverage of GC-rich regions of the genome. This was observed for DNA extracted from blood, cell lines, and formalin-fixed paraffin embedded (FFPE) tumor samples. More experiments are underway to determine the effect of the improved recovery of high-GC content DNA fragments on CNV detection.

Citation Format: Aaron R. Thorner, Ashwini Sunkavalli, Ling Lin, Robert T. Jones, Laura Schubert, Matthew D. Ducar, Ravali Adusumilli, Deniz Nesli Dolcen, Liuda Ziaugra, Jack Lepine, Laura E. MacConaill, William C. Hahn, Matthew Meyerson, Paul Van Hummelen. Reducing GC-bias and improving coverage distribution in Illumina sequencing using the Kapa Biosystems library construction method. [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 3583. doi:10.1158/1538-7445.AM2014-3583
Abstract 3586: HIF-1α from tumor-surround fibroblasts contributes to breast cancer malignant progression
Authors: Lu Yi , Zhang Jun
Published: May 2015

As tumor-surrounding stromal cells (such as fibroblasts) are integrate components of the tumor environment that together as an entity plays critical roles in the tumor progression, we investigated whether HIF-1α from tumor-surrounding fibroblasts plays a critical role in breast cancer (BCa) malignant progression. We co-cultured BCa 4T1/GFP cells with MEF fibroblasts that either expressed or lacked the HIF-1α gene in a 3D culture system established in our lab. The mixed cells (BCa:MEF) grew in a suspension manner and formed aggregates/spheroids. We found that HIF-1α plays a role under hypoxia in the 3D BCa cells' protrusion formation, an established marker for invasive, aggressively malignant tumor cells that start to invade the surrounding matrix. These protrusions (fluorescent) only appeared in mixed-cell aggregates containing HIF-1α wild-type MEF fibroblasts; but not appear in mixed-cell aggregates containing HIF-1α knockout MEF fibroblasts, implying HIF-1α from fibroblasts (MEF) plays a critical role in inducing surrounding epithelial BCa cells to form protrusions, an indicator that tumor cells have become invasive. Moreover, we performed transwell overnight invasion assay of BCa LM2 cells with and without cocultured MEF under both normoxia (21% O2) and hypoxia (1% O2). We found that MEF significantly promoted BCa invade through matrigel, and HIF-1α WT MEF promoted BCa invasion more than HIF-KO MEF did under hypoxia. Furthermore, by using Boyden's chamber-based cell migration assay, we found that migration of HIF-1α WT BCa (MMTC-PyT) cells increased more than two fold under hypoxia than normoxia. However, this hypoxia-induced increase in migration disappeared if HIF-1α KO counterparts were used. The loss of this hypoxia-induced migration in HIF-1α KO cells can be rescued by exogenous HIF-1α with Ad-HIF-1α transduction. A similar pattern of HIF-1α-dependent, hypoxia-stimulated cell migration was observed in fibroblasts when we analyzed HIF-1α WT and KO MEF cells, demonstrating that hypoxia stimulated a HIF-1α-dependent cell migration in both BCa and MEF cells. Together, these results indicate that HIF-1α from tumor-surrounding fibroblasts plays critical roles in promoting BCa malignant progression.

Citation Format: Yi Lu, Jun Zhang. HIF-1α from tumor-surround fibroblasts contributes to breast cancer malignant progression. [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 3586. doi:10.1158/1538-7445.AM2014-3586
Abstract 3588: Reactivation of dormant estrogen-dependent breast cancer micrometastases by bone marrow stromal injury in an in vitro model of dormancy
Authors: Tivari Samir , Lu Haiyan , Dasgupta Tanya , Wieder Robert
Published: May 2015

Estrogen-dependent (ER+) breast cancer can remain dormant in the bone marrow for more than a decade and continues to recur in a stochastic pattern at continuous, low rates. Mechanisms of dormancy in the bone marrow are mostly uncharacterized but data suggest that dormancy depends on close interactions of micrometastases with stromal elements and on effects by structural and soluble proteins. We have developed an extensive and well characterized in vitro model incorporating relevant elements of the bone marrow niche. We have demonstrated that FGF-2, abundantly deposited on bone marrow stroma, induces a partial re-differentiation of ER+ human breast cancer cell lines, manifested in part by cell cycle arrest in G1 and re-expression of integrins lost with dedifferentiation. Ligation of integrin α5β1 by incubation of cells on fibronectin completes the model. It specifically induces a dormant epithelial phenotype with RhoA inactivation through the Rho Gap GRAF, cortical actin rearrangement, omnidirectional focal complex activation and adhesion and activation of survival signaling. The signaling is dually initiated through FGF-2 receptors and fibronectin ligation, which activate PI3K/Akt, Erk and p38. The model is recapitulated on both human and murine bone stromal monolayer coincubations of human ER+ breast cancer cell lines, with the FGF-2 and fibronectin provided by the stroma. Mechanisms of breast cancer recurrence are also not understood, but data in other systems suggest that inflammatory cytokines can stimulate proliferation of breast cancer cells. We hypothesized that injury to bone marrow stroma can be the source of such cytokines. Towards this end, we injured human and murine bone marrow stromal monolayers by oxidation and hypoxia through one hour exposures to variable concentrations of H2O2 and a cyanide-containing analog, respectively, and by estrogen deprivation. These interventions induced secretion of IL-6 and IL-8, and induced activation TGFβ and TNFα under variable conditions. Incubation of human recombinant IL-6, IL-8 and TGFβ with dormant colonies in fibronectin culture induced dormant clones to proliferate into growing colonies. These data suggest a potential mechanism for reactivation of dormant breast cancer micrometastases and identify potential targets for suppression of the effect.

Citation Format: Samir Tivari, Haiyan Lu, Tanya Dasgupta, Robert Wieder. Reactivation of dormant estrogen-dependent breast cancer micrometastases by bone marrow stromal injury in an in vitro model of dormancy. [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 3588. doi:10.1158/1538-7445.AM2014-3588
Abstract 3589: Adipose stem cell-derived interferon gamma alters breast epithelial stem cell hierarchy
Authors: Anjanappa Manjushree , Burnett Riesa M. , Merfeld-Clauss Stephanie , Wooden William A. , March Keith L. , Tholpady Sunil , Nakshatri Harikrishna
Published: May 2015

Autologous adipose stem cell transplantation is increasingly being used for soft tissue reconstruction, particularly after mastectomy. However, a recent study demonstrated recurrence of invasive ductal carcinoma in the breast within four months of autologous fat grafting. Previous studies have documented how cancer cells commandeer adipose stem cells (ASC) to support their survival by altering extracellular matrix composition and stiffness. This study focused on delineating the effects of ASCs on the self-renewal capacity of breast epithelial cells. The immortalized breast epithelial cell line MCF-10A, MCF-10A overexpressing Slug, and MCF-10A overexpressing SRC were examined using a mammosphere assay and flow cytometry for the effects of ASCs on their self-renewal and stem-luminal progenitor-differentiated cell surface marker profile. MCF-10A-Slug cells provided a unique model system to study breast epithelial cell dedifferentiation as Slug functions downstream of the breast cancer susceptibility gene BRCA1 and is a major suppressor of progenitor cell lineage commitment and differentiation. Interestingly, results with these established cell lines were reproduced in primary breast epithelial cells isolated from a high-risk patient. We show that secreted factors from ASCs promote self-renewal of all cell types examined. ASC co-culture or treatment with ASC conditioned media (CM) altered the number of CD49fhigh/EpCAMlow stem-like and CD49fmedium/EpCAMmedium luminal progenitor cells in 2D culture and mammosphere assays. ASCs secrete Interferon gamma (IFNγ), CD40 ligand, IL-23 and SERPINE1. Among these factors, only IFNγ treatment altered stem cell hierarchy; while MCF-10A-Slug cells displayed only luminal progenitor phenotype, cells treated with IFNγ contained a distinct stem-like cell population. These results suggest that paracrine activity of adipose stem cell-derived factors can either enhance dedifferentiation and promote proliferation of dormant tumor cells or predispose normal cells to transformation by blocking differentiation.

Financial support: Susan G. Komen For the Cure SAC110025 and Zeta Tau Sorority (HN)

Citation Format: Manjushree Anjanappa, Riesa M. Burnett, Stephanie Merfeld-Clauss, William A. Wooden, Keith L. March, Sunil Tholpady, Harikrishna Nakshatri. Adipose stem cell-derived interferon gamma alters breast epithelial stem cell hierarchy. [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 3589. doi:10.1158/1538-7445.AM2014-3589
Abstract 3592: Macrophages confer resistance to cisplatin in MTLn3 and MDA-MB 231 breast cancer cells
Authors: Sabari Joshua K. , Cabrera Ramon , Segall Jeffrey
Published: May 2015

Drug resistance presents a clinical challenge to the treatment of breast cancer. Stromal cells such as macrophages may play a role in drug resistance. The purpose of this study is to examine the interaction between breast cancer cells and macrophages and its effects on drug therapy resistance. The five-year survival for localized hormone sensitive disease is 98% and as low as 15% for triple negative (ER/PR/Her2) and metastatic disease. Most targeted anticancer therapies, as well as cytotoxic therapies, are burdened by the development of cancer cell resistance. It is hypothesized that the tumor microenvironment may confer innate resistance to drug therapy. It is well established that chemotactic factors found in the tumor microenvironment can promote motility and invasion. We hypothesize that tumor associated macrophages can confer drug resistance to breast cancer cells via overexpression of epidermal growth factor (EGF). EGF is overexpressed in many tumor types and is clinically correlated with a poor overall prognosis. Previous studies have shown that EGF is produced by non-tumor cells in the microenvironment; in particular tumor-associated macrophages (TAM) and endothelial cells. We have previously shown that macrophages and tumor cells, via a paracrine loop, are necessary and sufficient for comigration and invasion. Breast cancer cells produce colony stimulating factor 1 (CSF-1) which positively feeds back to increase macrophage expression of EGF. Increased EGF expression in turn leads to tumor growth and invasion. We selected two triple negative breast cancer cell lines, MDA-MB 231 (human mammary adenocarcinoma) and MTLn3 (rat mammary adenocarcinoma) that were transfected to express GFP. Co-culture cell survival assays were performed with macrophages, fibroblasts, and endothelial cells. All tissue culture was completed in DMEM (HyClone) plus 10% FBS. Cells were grown for 24 hours in co-culture, treated with chemotherapy (doxorubicin or cisplatin at a range of concentrations) for 24 hours, and then analyzed at 72 hours using the DXP-10 Calibur analyzer. Prior to analysis, cells were again replated to measure “replating efficiency” i.e. chemoresistant cell survival. We found that coculture with macrophages increased tumor cell survival upon exposure to cisplatin. We conclude that macrophages are an important component of the tumor microenvironment that may play a role in chemotherapy resistance.

Citation Format: Joshua K. Sabari, Ramon Cabrera, Jeffrey Segall. Macrophages confer resistance to cisplatin in MTLn3 and MDA-MB 231 breast cancer 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 3592. doi:10.1158/1538-7445.AM2014-3592
Abstract 3598: Macrophage-derived reactive oxygen species suppress miR-328 targeting CD44 in gastrointestinal cancer cells and promote redox adaptation
Authors: Ishimoto Takatsugu , Yoshida Naoya , Sugihara Hidetaka , Izumi Daisuke , Miyake Keisuke , Sawayama Hiroshi , Imamura Yu , Iwagami Shiro , Baba Yoshifumi , Baba Hideo
Published: May 2015

Background: CD44 has recently been identified as one of the cell surface markers associated with cancer stem cells (CSCs) in several types of tumor. We have reported the functional role of CD44 variant isoform in the maintenance of low reactive oxygen species (ROS) levels in gastrointestinal cancer cells. While, microRNA (miRNA) is involved in the pathogenesis of many cancers and the networks of miRNA regulate CSCs properties. However, the mechanism underlying the miRNA in the regulation of CSC marker is mostly unrevealed. Objectives: The aim of this study was to investigate the regulation of CD44 expression by miRNAs and to develop newmolecular targets in gastrointestinal cancer. Methods and Results: To investigate the mechanism in the regulation of CD44, We performed miRNA screening in six human gastrointestinal cancer cell lines and identified three candidate miRNAs that could regulate CD44 expression in gastrointestinal cancer. Among these, we focused on miR-328 and examined its functional relevance using growth assays and cytotoxicity assays. CD44 expression was reduced in gastrointestinal cancer cell lines forced to express miR-328, leading to inhibition of cancer cell growth in vitro and in vivo, and impaired resistance to chemotherapeutic drugs and reactive oxygen species (ROS). In contrast, induction of CD44 expression by miR-328 inhibitor led to promotion of cancer cell growth. Furthermore, we revealed that ROS produced by macrophages triggered CD44 expression through suppression of miR-328 in gastric cancer cells. Finally, tumor-infiltrating macrophages (CD68 and CD163) were closely related to both miR-328 down-regulation and CD44 up-regulation in 63 patients with surgically-resected gastric cancer. Conclusions: Macrophages in the tumor microenvironment may cause increased CD44 expression through miR-328 suppression, resulting in tumor progression by enhancing ROS defense. miR-328-CD44 signaling mediated by macrophages may thus represent a potential target for the treatment of gastrointestinal cancer.

Citation Format: Takatsugu Ishimoto, Naoya Yoshida, Hidetaka Sugihara, Daisuke Izumi, Keisuke Miyake, Hiroshi Sawayama, Yu Imamura, Shiro Iwagami, Yoshifumi Baba, Hideo Baba. Macrophage-derived reactive oxygen species suppress miR-328 targeting CD44 in gastrointestinal cancer cells and promote redox adaptation. [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 3598. doi:10.1158/1538-7445.AM2014-3598
Abstract 3601: Stromal fibroblasts mediate extracellular matrix remodeling and invasion of scirrhous gastric carcinoma cells
Authors: Yamaguchi Hideki , Yanagihara Kazuyoshi , Yashiro Masakazu , Sakai Ryuichi
Published: May 2015

Scirrhous gastric carcinoma (SGC; also called diffusely infiltrative carcinoma, Borrmann's type-IV carcinoma, or the linitis plastica-type carcinoma) has the worst prognosis of all gastric cancers, owing to its rapid expansion by invasion and frequent peritoneal dissemination. Due to the increased proliferation of stromal fibroblasts (SFs) that occurs within SGC lesions and the peritoneal metastatic sites, SFs have been proposed to support the progression of this disease. However, the biological and molecular basis and the pathological role of the intercellular interaction between SGC cells and SFs remain largely unknown. In this study, we investigated the role of SFs in the invasion of the extracellular matrix (ECM) by SGC cells. When SGC cells were cocultured with SFs derived from SGC tissue on three-dimensional (3D) Matrigel, they were attracted together to form large cellular aggregates that invaded within the Matrigel. Time-lapse imaging revealed that this process was associated with extensive contraction and remodeling of the ECM. Immunofluorescence and biochemical analysis showed that SGC cells stimulate phosphorylation of myosin light chain and actomyosin-mediated mechanical remodeling of the ECM by SFs. By utilizing this assay system for inhibitor library screening, we have identified several inhibitors that potently suppress the cooperation between SGC cells and SFs to form the invasive structures. Among them, a Src inhibitor dasatinib impaired the interaction between SGC cells and SFs both in vitro and in vivo and effectively blocked peritoneal dissemination of SGC cells. These results indicate that SFs mediate mechanical remodeling of the ECM by SGC cells, thereby promoting invasion and peritoneal dissemination of SGC.

Citation Format: Hideki Yamaguchi, Kazuyoshi Yanagihara, Masakazu Yashiro, Ryuichi Sakai. Stromal fibroblasts mediate extracellular matrix remodeling and invasion of scirrhous gastric carcinoma 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 3601. doi:10.1158/1538-7445.AM2014-3601
Abstract 3602: PTEN dependent angiogenesis is mainly regulated by (tumor secreted-) uPAR
Authors: Unseld Matthias , Chilla Anastasia , Pausz Clemens , Breuss Johannes , Schabbauer Gernot , Prager Gerald
Published: May 2015

Tumor stroma crucially influences the growth of most epithelial tumors and is in focus of many therapeutic approaches. Several oncogenes and tumor suppressors coordinate complex signaling cascades in the tumor and its microenvironment. In this context, downregulation of the tumor suppressor PTEN was shown to be indispensable for (tumor-)angiogenesis and cancer outgrowth. However, the essential process leading to PTEN downregulation, favouring a pro-angiogenic phenotype, in tumor vasculature is unknown so far. Here we show that PTEN-dependent angiogenesis is centrally affected by tumor- secreted soluble uPAR. In detail, the presence of uPAR significantly influenced the levels of PTEN in vitro and in vivo. Endothelial cell stimulation with tumor- derived soluble uPAR remarkable decreased PTEN levels, which led to increased cell migration, enhanced wound healing as well as capillary-sprouting in vitro and enhanced endothelial cell invasion and functional vessel formation in a directed-in vivo angiogenesis assay. uPAR- derived PTEN regulation was not mediated via mRNA stability or decrease in PTEN degradation, but via regulation of PTEN transcription as revealed by actinomycin D experiments. uPAR thereby interacted with αv integrins, which enhanced FAK activation and - as a consequence - led to NFkB-dependent PTEN repression. Finally, the biological relevance of uPAR- dependent PTEN regulation was evaluated and confirmed via uPAR -/- crossbreds with either PTEN +/- or PTEN +/+ mice. Our results provide first evidence of the role of tumor-derived uPAR in regulation of the central phosphatase PTEN in angiogenic cell behaviour. Our findings give novel insights into the complex mechanism of the tumor- microenvironmental interaction and might lead to new therapeutic strategies in cancer.

Citation Format: Matthias Unseld, Anastasia Chilla, Clemens Pausz, Johannes Breuss, Gernot Schabbauer, Gerald Prager. PTEN dependent angiogenesis is mainly regulated by (tumor secreted-) uPAR. [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 3602. doi:10.1158/1538-7445.AM2014-3602
Abstract 3605: The inhibitor of NF-ĸB kinase, IKKβ, regulates the stability of GLI1 transcription factor
Authors: Agarwal Nitin K. , Kunkalla Kranthi , Vega Francisco
Published: May 2015

Lymphoma microenvironment has a crucial role in lymphoma initiation, progression and drug resistance. Fibroblastic reticular cells (FRCs) participate dynamically in the cytokine microenvironment of the lymph node paracortex as well as the regulation of cell trafficking and access of T-cells into the paracortex. The FRC niche also participates in the cytokine microenvironment of lymphomas providing homing and survival signals including CCL19, CCL21, IL-7, CXCL12 and hedgehog (Hh) ligands. Hh signaling is evolutionary conserved signaling pathway that serves several physiological and development processes mediated by externally secreted Hh ligands. We previously demonstrated that the canonical Hh ligand-PTCH1-SMO-GLI axis is functional and contributes to cell survival, proliferation and enhances chemo tolerance in DLBCL. We further showed that GLI1, Hh signaling transcription factor is aberrantly activated in ∼87% of DLBCL and this activation is in part mediated by sonic Hh ligands secreted by stromal cells. Although the importance of GLI1 in tumor development is well recognized, the molecular mechanisms controlling the transcriptional activity of GLI1 is limited. To identify regulatory components that participate in the transcriptional activity of GLI1, we explored GLI1 putative interacting proteins by liquid chromatography tandem mass spectrometry following immunoprecipitation of endogenous GLI1. We identified the inhibitor of NF-ĸB kinase, IKKβ as a novel GLI1-binding protein. In addition, we found that the kinase activity of IKKβ is critical to recruit and phosphorylate GLI1 in the C-terminal fragment. We further showed that in DLBCL cells stimulated with TNFα, IKKβ phosphorylates GLI1 and restricts the binding between GLI1 with HECT-type E3 ubiquitin ligase (ITCH) resulting in GLI1 stability and increased oncogenic potential. Inhibition of IKKβ-mediated GLI1 phosphorylation restored the binding between GLI1 and ITCH and promoted ubiquitination and degradation of GLI1. Collectively, these results indicate that IKKβ regulates the transcriptional activity of GLI1 by phosphorylating GLI1 and modulating the binding between GLI1 and ITCH. In DLBCL, canonical NF-κB and GLI1 mediated Hh signaling pathways are strongly responsive with tumor microenvironment; our study will help to understand the cross talk between these two pathways which could represents a novel therapeutic approach for DLBCL.

Citation Format: Nitin K. Agarwal, Kranthi Kunkalla, Francisco Vega. The inhibitor of NF-ĸB kinase, IKKβ, regulates the stability of GLI1 transcription factor. [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 3605. doi:10.1158/1538-7445.AM2014-3605
Abstract 3607: Modulation of neuroblastoma metabolism by an extracellular chaperone SPARC
Authors: Chlenski Alexandre , Dobratic Marija , Salwen Helen R. , Applebaum Mark , Cohn Susan L.
Published: May 2015

Secreted Protein Acidic and Rich in Cysteine (SPARC) is an extracellular matrix protein with diverse biologic functions. We have shown that SPARC is a scavenger chaperone that regulates matrix degradation, remodeling, and repair. We have also demonstrated that SPARC blocks angiogenesis and inhibits the growth of several cancers, including neuroblastoma. In this study we examined how extracellular chaperone SPARC affects neuroblastoma tumor progression.

The chaperone properties of SPARC were characterized with cell biology methods and Surface Plasmon Resonance. Neuroblastoma cell lines derived from the high-risk tumors were treated with SPARC. The effect of SPARC on cancer cell metabolism was characterized using the Seahorse analyzer with and without inhibitors of glycolysis and mitochondrial oxidation. Standard cell culture techniques were used to assess the effect of SPARC-induced metabolic changes on neuroblastoma cell proliferation and survival.

We demonstrate that SPARC functions as a chaperone, shuttling extracellular ligands between cellular and extracellular tissue compartments. We characterize physical and biological properties of SPARC-mediated ligand transport, and further demonstrate that as a chaperone, SPARC can significantly affect tissue physiology, to the degree of altering cell metabolism. We show that in addition to inhibition of neuroblastoma progression in vivo, SPARC-induced changes in cellular metabolism severely impair neuroblastoma growth and survival in vitro.

This novel mechanism of metabolism regulation by an extracellular chaperone SPARC may lead to additional insights into the therapy of neuroblastoma.

Citation Format: Alexandre Chlenski, Marija Dobratic, Helen R. Salwen, Mark Applebaum, Susan L. Cohn. Modulation of neuroblastoma metabolism by an extracellular chaperone SPARC. [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 3607. doi:10.1158/1538-7445.AM2014-3607
Abstract 3610: High stromal S100A9 expression serves an early oral cancer recurrence marker possibly through enhancing cancer cell invasivenes, angiogenesis, and differentially recruiting infiltrating immune cells
Authors: Wu Li-Wha
Published: May 2015

S100A9 belongs to S100 family with Ca+2-binding EF-hands in both N- and C-termini and is frequently deregulated in several cancer types. Consistent with this notion, we identified the increase of S100A9 mRNA expression by 2.6 fold (p<0.01) in oral cancer cells when compared to normal oral keratinocytes using DNA microarray analysis. The increase of S100A9 protein validated in clinical tissue specimens was also detected in the adjacent tumor stroma, especially in CD15+ or CD68+ immune infiltrates. High S100A9 expression in the stroma was positively associated not only with the poor differentiation of tumor but also tumor recurrence. Moreover, high stromal S100A9 expression in early-stage patients had shorter recurrence-free survival, suggesting a role of S100A9 in oral cancer recurrence. Although altering the expression of S100A9 affected cell proliferation depending on the cell lines, S100A9 functioned as a promoting molecule for cell migration and invasion. Moreover, S100A9 secreted from oral cancer cells inhibited anti-inflammatory IL-10 production and acted as positive regulator in angiogenesis. Ectopic S100A9 expression promoted subcutaneous tumorigenesis in vivo with the enhanced recruitment of a CD11c+ myeloid cells and production of several inflammatory cytokines. The serum concentration of S100A9, a secretable protein, was significantly increased in early-stage but not late stage oral cancer patients. Together, stromal S100A9 plays a tumor-promoting role in oral carcinogenesis and serum S100A9 may serve as an early marker for cancer recurrence. Understanding the potential role of S100A9 in oral cancer and stromal cells should facilitate the development of new therapeutic approaches for oral cancer treatment.

Citation Format: Li-Wha Wu. High stromal S100A9 expression serves an early oral cancer recurrence marker possibly through enhancing cancer cell invasivenes, angiogenesis, and differentially recruiting infiltrating immune 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 3610. doi:10.1158/1538-7445.AM2014-3610
Abstract 3611: Stromal transforming growth factor-beta 1 is crucial for reinforcing the invasive potential of low invasive cancer
Authors: Hwang Young Sun , Park Kwang-Kyun , Chung Won-Yoon
Published: May 2015

Comparative analyses of gene expression profiles between metastatic and non-metastatic cells have revealed that various genes are differentially expressed in association with the metastatic potential of cancer cells. Interactions between tumor cells and their adjacent microenvironment are intimately associated with tumor growth and the development of metastasis. Cancer-associated fibroblasts (CAFs) are a source of various paracrine and autocrine growth factors that regulate growth of surrounding cells as well as themselves. Thus, there is an increasing interest to study CAFs as drug targets for anticancer therapies.

In the present study, we investigated the existence of molecular crosstalk between cancer cells and surrounding stromal cells to reinforce the invasive potential of low invasive cancer. Molecular crosstalk between cancer cells and tumor microenvironment components suggests potential targets for new therapeutic approaches.

Material and methods: Isolated normal fibroblasts (NFs) or CAF was co-cultured with low invasive HSC-2 cells to evaluate whether stromal TGF-β1 induced PDPN expression by Transwell invasion and influenced tumor growth in orthotopic xenografts.

Results: Stimulation by TGF-β1 promoted PDPN expression and Transwell invasion through SMAD signaling as well as activation of Src, P38 mitogen activated protein kinase, and extracellular regulated kinase 1/2. PDPN induction was TβRII-dependent. Tumor growth of HSC-2 OSCC in a mouse orthotopic xenograft was intensified in the tumor CAF microenvironment.

Conclusions: Stromal TGF-β1 signaling promoted PDPN expression in cancer cells, thereby enhancing tumor growth and leading to a more invasive phenotype.

Citation Format: Young Sun Hwang, Kwang-Kyun Park, Won-Yoon Chung. Stromal transforming growth factor-beta 1 is crucial for reinforcing the invasive potential of low invasive 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 3611. doi:10.1158/1538-7445.AM2014-3611
Abstract 3612: Effect of cancer associated fibroblast on gastric cancer cells under hypoxia
Authors: Kinoshita Haruhito , Yashiro Masakazu , Masuda Go , Kasashima Hiroaki , Morisaki Tamami , Fukuoka Tatsunari , Shibutani Masatune , Yamazoe Sadaaki , Sakurai Katsunobu , Nagahara Hisashi , Kimura Kenjiro , Toyokawa Takahiro , Amano Ryosuke , Kubo Naoshi , Tanaka Hiroaki , Muguruma Kazuya , Otani Hiroshi , Maeda Kiyoshi , Ohira Masaichi , Hirakawa Kosei
Published: May 2015

Background:

Cancer-associated fibroblasts (CAF) have been suggested to be associated with the scirrhous gastric cancer (SGC) progression. SGC have a heterogeneously hypoxic environment which has been currently considered to be associated with aggressive tumor phenotypes cancer. Stromal cell-derived factor (SDF)-1 and C-X-C chemokine receptor type 4 (CXCR4) form an important chemokine/receptor pair, and are supposed to be up-regulated by hypoxia inducible factor-1 (HIF-1). The aim of our study was to clarify the effect of hypoxic environment on the CAF and CXCR4/SDF-1 axis in SGC.

Experimental Design:

Proliferation of SGC cells grown in monoculture and co-culture with CAF under hypoxia. SGC and CAF expression level of CXCR4 and SDF-1 under hypoxia were examined by RT-PCR and ELISA. Proliferation and migration of SGC cells with SDF-1 or hepatocyte growth factor (HGF) or conditioned medium from CAF under hypoxia, furthermore, added c-Met inhibitor or CXCR4 inhibitor.

Results:

The proliferation that co-culture of SGC cells with CAF was significantly increased under hypoxia. The expression level of CXCR4 mRNA was significantly increased under hypoxia in all of SGC cells. SDF-1 concentration level was significantly increased under hypoxia. The proliferation of SGC cells with SDF-1 and CAF was significantly increased. The proliferation of SGC cells with CAF was inhibited by CXCR4 inhibitor under hypoxia. The migration of SGC cells with HGF and CAF was significantly increased. The migration of SGC cells with CAF was inhibited by c-Met inhibitor.

Conclusion:

CAF might up-regulate the proliferation and migration of SGC cells under hypoxia, in comparison with that under normoxia. CXCR4/SDF-1 axis might play an important role for the proliferation of SGC cells under hypoxia.

Citation Format: Haruhito Kinoshita, Masakazu Yashiro, Go Masuda, Hiroaki Kasashima, Tamami Morisaki, Tatsunari Fukuoka, Masatune Shibutani, Sadaaki Yamazoe, Katsunobu Sakurai, Hisashi Nagahara, Kenjiro Kimura, Takahiro Toyokawa, Ryosuke Amano, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Hiroshi Otani, Kiyoshi Maeda, Masaichi Ohira, Kosei Hirakawa. Effect of cancer associated fibroblast on gastric cancer cells under hypoxia. [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 3612. doi:10.1158/1538-7445.AM2014-3612
Abstract 3640: IL-9 is involved in the establishment of a tolerogenic milieu that prevents anti-tumor immunity
Authors: Hoelzinger Dominique B. , Dominguez Ana Lucia , Cohen Peter A. , Gendker Sandra J.
Published: May 2015

IL-9 is a Th2 cytokine associated with T regulatory cell function, parasite expulsion and allergic airway inflammation. In many tumors IL-9 contributes to the establishment of a tolerogenic / immunosuppressive environment or acts directly to drive tumor growth, whereas in melanomas it inhibits tumor growth. Little is known about the effect of IL-9 in breast cancer biology. Here we show that IL-9 is a key factor in establishing a permissive growth environment for two murine breast cancer cells lines: TUBO cells that express Her2/neu and 4T1 cells that resemble aggressive, triple-negative breast cancers. We observed that TUBO cells were rejected by 78%, and 4T1 cells by 68% of IL-9 -/- (IL-9ko) mice, and that in the remaining mice the tumors developed later and grew slower than in WT mice. Tumor rejection is CD8+ T cell dependent and is capable of producing a memory response against tumor rechallenge. Delayed depletion of CD8+ T cells in mice that had rejected 4T1 cells showed no tumor growth in 63 % of the mice, suggesting that in these mice the tumor had been completely eradicated and was not held in stasis by immune surveillance. Furthermore we found a 3.7 fold increase (p=0.007) in the number of activated CD8+ T cells derived from IL-9ko mice rejecting tumors as compared to CD8+ T cells from tumor bearing WT mice. More importantly, the transfer of activated splenocytes from IL-9ko mice led to tumor rejection in WT mice (p<0.001). Moreover, WT mice treated with neutralizing anti-IL-9 evidenced slower 4T1 tumor growth compared to untreated and isotype control antibody treated mice. These data suggest that IL-9 neutralization should be examined as an adjuvant to immunotherapeutic or chemotherapeutic approaches.

Supported by the NCI : CA155295

Citation Format: Dominique B. Hoelzinger, Ana Lucia Dominguez, Peter A. Cohen, Sandra J. Gendker. IL-9 is involved in the establishment of a tolerogenic milieu that prevents anti-tumor immunity. [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 3640. doi:10.1158/1538-7445.AM2014-3640
Abstract 3642: Cancer therapy by resuscitating Notch immune surveillance
Authors: Dudimah Duafalia F. , Pellom Samuel T. , Uzhachenko Roman V. , Carbone David P. , Dikov Mikhail M. , Shanker Anil
Published: May 2015

The immunosuppressive tumor microenvironment perturbs numerous immune regulatory networks and usurps host anti-tumor immunity. We discovered that tumor interferes with host hematopoietic Notch system in lung cancer patients. The resultant decrease in immune Notch signaling could be a major causative link in the inadequate induction of anti-tumor immunity. Interestingly, we found that tumor-induced decrease in immune Notch in various mouse solid tumor models could be restored therapeutically by the following two agents. Administration of a novel Delta-like ligand 1 (DLL1) multivalent cluster and the FDA-approved proteasome inhibitor drug bortezomib - which also sensitizes tumors to death signals - could activate Notch 1 signaling in lymphoid cells of tumor-bearing mice without increasing tumor cell proliferation or clonogenicity. Systemic activation of DLL1-Notch signaling could attenuate tumor vascularization as well as increase T cell infiltration in tumor, decrease proportion of regulatory T cells and enhance antitumor T cell function and memory in multiple mouse tumor models. New data also show that bortezomib affects the expression of notch receptors and ligands differentially in lymphocytes and in a wide range of solid tumor cells. Moreover, bortezomib administration increased the expression of Notch target genes Hes 1 and Hey 1 in thymus, lymph node, and spleen, as well as decreased the proportion of regulatory T cells and enhanced T cell production of IFN-γ in tumor-bearing mice. Results indicate that bortezomib-induced activation of Notch target genes Hes 1 and Hey 1 is through its inhibition of NFkB while its activation of another Notch target gene Deltex 1 is mediated via PI3K. The findings suggest a potential synergistic action of bortezomib and DLL1 activation of Notch signaling. The potential of modulating anti-tumor Notch signaling by the prototypic DLL1 cluster in combination with bortezomib presents exciting opportunities to uncover multi-pronged immune stimulatory regimens. Therapeutic restoration of immune Notch signaling could provide effective treatment and recurrence-free survival in cancer patients by breaking tumor resistance, enhancing immune surveillance, and sustaining robust anti-tumor immunity.

Citation Format: Duafalia F. Dudimah, Samuel T. Pellom Jr., Roman V. Uzhachenko, David P. Carbone, Mikhail M. Dikov, Anil Shanker. Cancer therapy by resuscitating Notch immune surveillance. [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 3642. doi:10.1158/1538-7445.AM2014-3642
Abstract 3648: Expression of tolerogenic enzymes IDO-1, IDO-2 and TDO in commonly used mouse tumor models and impact on model selection for evaluation of immunosuppression reversal by novel therapeutics
Authors: Noubade Rajkumar , Kohrt Holbrook , Marshall Lisa , Sagiv-Barfi Idit , Hebb Jonathan , Chester Cariad , Rajapaksa Amanda , Waller Erin , Young Steve , Powers Jay , Jaen Juan
Published: May 2015

Objective:

Inhibition of indoleamine-2,3-dioxygenase-1 (IDO-1), indoleamine-2,3- dioxygenase-2 (IDO-2), and/or tryptophan-2,3-dioxygenase (TDO) represent novel opportunities for reversing the immunosuppressed microenvironment found in cancer patients. However, little is known about the relative expression of these enzymes in tumors and associated lymph nodes of mouse tumor models, including those previously utilized in the preclinical characterization of tool compounds or even clinical candidates that inhibit one of these enzymes. This report describes the systematic characterization of expression of these enzymes in several of the most commonly used mouse tumor models.

Study Design:

Tumor models evaluated included B16F10 melanoma, B16-GMCSF melanoma and PAN02 pancreatic in C57BL/6 mice, and 4T1 breast and CT26 colon in Balb/c mice. Cells were injected s.c. into the flanks of 8-week old female mice. In each model, tumors and draining lymph nodes were collected from separate cohorts of mice when the mean tumor volumes reached 100 and 1,000 mm3, respectively. Expression of the 3 enzymes of interest was assessed using a variety of techniques, including qRT-PCR, Western blot and FACS.

Results:

A highly complex picture arises from our study. Expression levels of each of the enzymes studied here were highly dependent on the choice of tumor cell, the tissue analyzed (tumor vs. lymph node), as well as the stage of growth of the tumor. In general, the most commonly expressed of the 3 enzymes was IDO-1, primarily on tumor cells and dendritic cells in the lymph nodes.

Conclusion:

It is critically important, in the preclinical evaluation of potential new drugs, to select mouse models that recapitulate specific and well-characterized elements of the signaling pathway targeted by those drugs. The results presented in this communication should be of particular interest to those investigators pursuing a therapeutic strategy of immunosuppression reversal.

Citation Format: Rajkumar Noubade, Holbrook Kohrt, Lisa Marshall, Idit Sagiv-Barfi, Jonathan Hebb, Cariad Chester, Amanda Rajapaksa, Erin Waller, Steve Young, Jay Powers, Juan Jaen. Expression of tolerogenic enzymes IDO-1, IDO-2 and TDO in commonly used mouse tumor models and impact on model selection for evaluation of immunosuppression reversal by novel therapeutics. [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 3648. doi:10.1158/1538-7445.AM2014-3648
Abstract 3650: PI3-kinase gamma controls the macrophage M1-M2 switch, thereby promoting tumor immunosuppression and progression
Authors: Kaneda Megan , Gorjestani Sara , Varner Judith A.
Published: May 2015

Innate immune cells (macrophages, monocytes and neutrophils) rapidly migrate to sites of tissue damage where they initially activate T cells (M1 classical response). These cells gradually transition to suppress T cell responses, while stimulating wound healing (M2 alternative response). In tumors, however, the normal immune response is chronically disrupted and an exaggerated immunosuppressive, wound healing, tumor-promoting (M2) response prevails. In order to identify the mechanisms regulating this M2 response in tumor inflammation and to develop novel therapeutics to control it, we set out to explore the role of macrophage signaling in control of the M1-M2 switch.

Using RNA seq analysis of gene expression patterns in M1 and M2 macrophages in vitro and gene expression analysis of myeloid cells in spontaneous and orthotopic mouse models of lung, pancreatic and breast cancer, we found that myeloid cell PI3Kinase gamma controls the switch between the M1-M2 phenotype in human and murine macrophages. PI3Kinase gamma signaling stimulates an immunosuppressive, wound healing gene expression program exemplified by expression of immunosuppressive and pro-angiogenic factors, such as Arginase1, TGFbeta1, PDGFBB, MMP9, and MMP13, and suppression of pro-inflammatory factors such as IL12, iNos, and interferon gamma. Blockade of this pathway by genetic or pharmacological inhibition of PI3Kgamma blocks this immunosuppressive, wound healing gene expression program and stimulates instead a T cell activating gene expression program as well as T cell mediated tumor cell killing. PI3Kgamma blockade inhibits tumor growth and metastasis in mouse models of lung, pancreatic and breast carcinoma. Our studies demonstrate the critical role of PI3Kgamma signaling in the control of the M1-M2 macrophage switch and demonstrate that this kinase is an important therapeutic target to suppress tumor growth by targeting inflammation that is associated with all solid tumors.

Citation Format: Megan Kaneda, Sara Gorjestani, Judith A. Varner. PI3-kinase gamma controls the macrophage M1-M2 switch, thereby promoting tumor immunosuppression and progression. [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 3650. doi:10.1158/1538-7445.AM2014-3650