bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C25: Hsp27 is required for EGF-induced epithelial to mesenchymal transition in prostate cancer
Authors: Cordonnier Thomas , Bishop Jennifer L. , Shiota Masaki , Takeuchi Ario , Nip Ka Mun , Gleave Martin , Zoubeidi Amina
Published: August 2014

Prostate cancer (PCa) is the most common cancer and the second leading cause cancer death in North American men. Understanding the molecular mechanisms underlying metastatic PCa development is essential to decrease morbidity and mortality. An important determinant for metastasis is epithelial to mesenchymal transition (EMT). We show that the molecular chaperone Hsp27 drives EMT in PCa cells by decreasing epithelial cell markers, upregulating mesenchymal markers and enhancing cell migration and MMP-9 activity. Mechanistically, Hsp27 is required for EMT induced by epidermal growh factor (EGF) via modulation of the beta-catenin/Slug signaling pathway. Hsp27 knockdown induces sequestration of beta-catenin in the cytoplasm by increasing its binding to GSK3beta and abrogating EGF induced beta-catenin nuclear translocation. Additionally, by decreasing the nuclear translocation of beta-catenin, Hsp27 knockdown decreases the binding of beta-catenin to the Slug promoter, thereby abrogating its transcriptional activity. As Slug is known to be a critical factor in EMT induction, we confirmed the role of Hsp27 in EGF mediated EMT using Hsp27 knockdown. To address a role for Hsp27 in promoting aggressive PCa in vivo, Hsp27 was suppressed in a murine model of PCa using the antisense drug OGX-427 (currently in phase II clinical trials). We found OGX-427 treatment significantly reduces tumor cell metastasis in mice, and moreover, decreases circulating tumor cell counts in patients with castration resistant prostate cancer. This study defines a role for Hsp27 as a critical regulator of EGF dependent EMT and highlights Hsp27 is an attractive anti-cancer target for metastatic PCa.

Citation Format: Thomas Cordonnier, Jennifer L. Bishop, Masaki Shiota, Ario Takeuchi, Ka Mun Nip, Martin Gleave, Amina Zoubeidi. Hsp27 is required for EGF-induced epithelial to mesenchymal transition in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C25.
Abstract C26: Molecular determinants of the intravasation step in cancer metastasis
Authors: Deryugina Elena I. , Quigley James P.
Published: August 2014

Intravasation, the directional entry of primary tumor cells into host vasculature, remains the most understudied step in the metastatic cascade, likely due to deficiency in models recapitulating the intravasation process and allowing for molecular probing and intervention in vivo. By employing chick embryo models of spontaneous metastasis, where human tumor cells form microtumors and disseminate into secondary tissues strictly via an angiogenic vasculature, we have identified several rate-limiting processes and critical molecules of tumor and host origin, which functionally contribute to intravasation. We have demonstrated that the induction of an intravasation-sustaining tumor microenvironment involves tumor-produced cytokines, including IL-8 that triggers the influx of neutrophils into developing primary tumors. These inflammatory neutrophils deliver a potent pro-angiogenic protease, TIMP-1-free MMP-9, which releases stroma-bound angiogenic factors such as FGF-2 and VEGF. In addition, our recent findings indicate that M2-polarized macrophages also produce TIMP-1-deficient MMP-9, which explains the high angiogenic potential of this sub-type of inflammatory cells. Whereas host inflammatory cell MMP-9 promotes tumor angiogenesis, one of the tumor-produced MMPs, namely MMP-1, appears to regulate the microarchitecture of intratumoral vasculature. We have demonstrated that carcinoma cell MMP-1 facilitates tumor cell intravasation by specific induction of vascular permeability via activation of PAR1 expressed on the angiogenic endothelial cells. Finally, we show that intimate interactions between tumor cells and host endothelial cells result in de novo generation of plasmin that cleaves a transmembrane molecule, CDCP1, regulating tumor cell survival and motility signaling, which allows for successful intravasation. Together, our findings illuminate how different proteolytic enzymes, implicated mostly in path-clearing matrix proteolysis and tumor invasion, also orchestrate localized protein modifications and regulate cellular processes leading to, and following the intratumoral intravasation events.

Citation Format: Elena I. Deryugina, James P. Quigley. Molecular determinants of the intravasation step in cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C26.
Abstract C37: Positive involvement of chemokine CXCL14 in osteolytic bone metastasis of lung cancer
Authors: Iguchi Haruo , Takiguchi Soichi
Published: August 2014

To investigate the molecular mechanisms for osteolytic bone metastasis, we established a human lung cancer cell line (HARA-B4) with elevated bone metastatic activity. All the experiments employed by HARA-B4 were performed with the parental HARA as a control cell line. Anchorage-independent growth ability was higher in HARA-B4 according to the results by soft agar colony formation assay. Murine macrophage-like and osteoblast-like cell lines (RAW264.7 and MC3T3-E1, respectively) migrated faster toward cultured HARA-B4 in transwell cell migration assay. The spans of bone metastatic emergence and survival were shortened in nude mice inoculated with HARA-B4. CXC chemokine ligand 14 (CXCL14) was overexpressed in HARA-B4 and responsible for the activities described above according to the results performed with the CXCL14 siRNA-treated HARA-B4. Furthermore, in the clinical specimens of the lung cancer-induced bone metastasis, expression of CXCL14 was observed in the infiltrated tumor cells of the bone marrow as well as bone stromal cells in all the specimens examined (n=7). These data indicates the positive involvement of CXCL14 in the bone metastasis from lung cancer.

Citation Format: Haruo Iguchi, Soichi Takiguchi. Positive involvement of chemokine CXCL14 in osteolytic bone metastasis of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C37.
Abstract C40: αvβ6 integrin promotes a TGFβ1-mediated cancer cell autonomous osteolytic program through upregulation of matrix metalloproteinase 2 (MMP2)
Authors: Dutta Anindita , Li Jing , Lu Huimin , Akech Jacqueline , Pratap Jitesh , Wang Tao , FitzGerald Thomas J. , Jiang Zhong , Violette Shelia M. , Stein Gary S. , Lian Jane B. , Languino Lucia R.
Published: August 2014

Metastatic prostate cancer to the bone is associated with considerable morbidity and mortality. Because the molecular basis of this process is not completely understood, current treatment is largely palliative, aiming at reducing severe bone pain, nerve compression and pathological fractures. The molecular circuitries controlling osseous prostate metastasis are known to depend on the activity of a pro-metastatic cytokine, TGFβ1, and of matrix metalloproteinases (MMPs). MMP2 is known to enhance prostate cancer-mediated bone degradation and has been reported to be induced by TGFβ1. Various studies show that the αvβ6 integrin activates and is induced by TGFβ1, thus potentiating TGFβ1 responses in the sites where it is expressed. However, a role for αvβ6 in regulating TGFβ1—induced MMP2 has not been described before. Here, we demonstrate that the αvβ6 integrin is upregulated in human prostate cancer bone metastasis and promotes osteolysis as observed by μ–computed tomography (μ–CT) in an in vivo model of metastatic bone disease. Using immunoblotting and real-time PCR analysis, we provide evidence that the αvβ6 integrin sustains TGFβ1—dependent upregulation of MMP2 mRNA and protein levels by associating with TGFβ receptor II. We also show that αvβ6 contributes to upregulation of MMP2 by TGFβ1 through a highly specific transcriptional program mediated by Smad3, and promotes osteolysis in vivo through upregulation of MMP2 in cancer cells. In conclusion, we have identified a novel role of the αvβ6 integrin in directing, in vivo, a cell-autonomous osteolytic program through activation of MMP2. These mechanistic studies will support the development of novel molecular strategies for prostate cancer bone metastasis through inhibition of the TGFβ1\αvβ6\MMP2 signaling pathway.

Acknowledgments: This work was supported by the following grants: NIH — RO1 CA89720 to Dr. Lucia R. Languino and NIH — PO1 CA140043 to Drs. Lucia R. Languino, Gary S. Stein, Jane B. Lian.

Drs. Dutta and Li share the first authorship.

Citation Format: Anindita Dutta, Jing Li, Huimin Lu, Jacqueline Akech, Jitesh Pratap, Tao Wang, Thomas J. FitzGerald, Zhong Jiang, Shelia M. Violette, Gary S. Stein, Jane B. Lian, Lucia R. Languino. αvβ6 integrin promotes a TGFβ1-mediated cancer cell autonomous osteolytic program through upregulation of matrix metalloproteinase 2 (MMP2). [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C40.
Abstract C50: CXCL12-triggered amoeboid cell motility is mediated through a RhoA-directed signaling hub
Authors: Wyse Meghan M. , Nestor-Kalinoski Andrea L. , Eisenmann Kathryn M.
Published: August 2014

Morphological plasticity is a common feature of cancer cells in response to environmental cues. Mesenchymal cancer cells, dependent on proteolysis for motility, can switch to a protease-independent amoeboid morphology and evade anti-invasion therapeutics that target matrix metalloproteases. The highly conserved formin family of proteins play a role in cell motility and play a critical role in mediating cancer cell invasion and metastasis. The cellular actin dynamics driving cell motility are highly regulated by mammalian diaphanous-related formin 2 (mDia2). mDia2 assembles non-branched actin filaments and maintains the cortical F-actin cytoskeleton. Our lab previously demonstrated a direct interaction between mDia2 and its inhibitor diaphanous-interacting protein (DIP), which subsequently acts as a trigger for mesenchymal/amoeboid interconvertability in breast cancer cells by disrupting F-actin polymerization and bundling. This interaction plays an essential role in the disruption of the cell cortex leading to a membrane blebbing phenotype, a hallmark of amoeboid cell motility (ACM). We recently reported that the chemokine CXCL12 stimulates DIP:mDia2 complex assembly. It is well established that CXCL12 and its receptor CXCR4 are involved in cancer metastasis, including breast cancer metastasis to lung and lymph node. Sites of distant metastasis are also often shown to express high levels of CXCL12. We, therefore, hypothesize that CXCL12 secreted from the tumor microenvironment may be a physiological trigger of amoeboid cell motility and invasion via the mDia2:DIP axis. Our current objective is to interrogate the requirement for CXCL12-driven, mDia2-dependent non-apoptotic membrane blebbing. Here we show in MDA-MB-231 human breast cancer cells that CXCL12 activates the GTPase RhoA, a known binding partner and activator of mDia2. Upon activation of mDia2, its inhibitor DIP can bind, disrupting F-actin polymerization within the cell cortex, leading to blebbing. Collectively, these data indicate a potential unexplored link between CXCL12/RhoA/mDia signaling and ACM in breast cancer cells. Understanding the molecular basis of ACM will lend novel insight into mechanisms controlling breast cancer progression and may translate into development of therapeutics controlling invasion/metastatic dissemination.

Citation Format: Meghan M. Wyse, Andrea L. Nestor-Kalinoski, Kathryn M. Eisenmann. CXCL12-triggered amoeboid cell motility is mediated through a RhoA-directed signaling hub. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C50.
Abstract C61: A macrophage autonomous α4β1integrin-Syk-Rac2 signaling axis controls macrophage differentiation, tumor growth, and metastasis
Authors: Joshi Shweta , Singh Alok Ranjan , Zulcic Muamera , Lei Bao , Messer Karen , Durden Donald L.
Published: August 2014

Macrophages, a major leukocyte population present in tumors, play an essential role in promoting tumor growth by affecting angiogenesis, immune suppression, invasion and metastasis. Despite considerable research efforts, the signal transduction events within macrophages which encode the complex cascade of events required for tumor growth and polarization of macrophages are poorly understood. Our previous findings demonstrate that α4β1 and αvβ3 integrin directed cell migration on specific extracellular matrices (ECM) requires a specific kinase-GTPase pair, Syk-Rac2. This pathway was shown to regulate the ECM dependent postnatal angiogenic response with no defect in vascular development. Herein, we have linked this ECM dependent signaling pathway in macrophages to the regulation of M2 macrophage differentiation and to the regulation of tumor growth, invasion and metastasis. Using mouse genetic models, we provide direct evidence that a macrophage specific, α4β1 integrin dependent Syk-Rac2 signaling axis acts in concert with the p110γ isoform (PTEN-PI-3 kinase pathway) to control HIF1α levels, tumor growth and metastasis. We report a novel proteasome/E3 ligase dependent mechanism by which PI-3 kinase regulates HIF1α levels under hypoxic conditions in the stromal compartment. Moreover, treatment with Syk or PI-3 kinase inhibitors demonstrate potent activity in multiple metastatic models. The results define a novel molecular mechanism for the regulation of HIF1α and a macrophage autonomous signaling pathway that is required for alterations in the ECM and the provisional integrin to regulate tumor metastasis and suggest treatment for metastatic disease targeting this pathway in the M2 macrophage compartment.

Citation Format: Shweta Joshi, Alok Ranjan Singh, Muamera Zulcic, Bao Lei, Karen Messer, Donald L. Durden. A macrophage autonomous α4β1integrin-Syk-Rac2 signaling axis controls macrophage differentiation, tumor growth, and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C61.
Abstract C84: Application of sensitive EMT reporter to identification of breast cancer stem cell-related genes
Authors: Yoshikawa Kiyotsugu , Matsumoto Yoshiaki , Ito Junji , Shiojiri Yoshie , Reinherz Ellis , Toi Masakazu
Published: August 2014

Epithelial-mesenchymal transition (EMT) is a key feature in breast cancer stem cell. In order to identify genes responsible for EMT/stemness, we have established a novel reporter system, in which mammary epithelial cells undergoing EMT can be visualized and positively selected by expression of GFP and blasticidine-resistant (BsR) genes, respectively. By incorporating tetracycline-responsive transactivator (rTA) and tetracycline-responsive element (TRE), vimentin promoter activity could be successfully amplified, which make it possible to induce sufficient Bsr/GFP fusion protein. Snail introduction can induce GFP+ CD44hi population by 30%. Using this reporter cell line, cDNA libraries derived from aggressive breast cancer cell lines MDA-MB-231 and BT549 were screened. Snail cDNA was isolated from several clones with GFP+ CD44hi. An shRNA library screening identified several candidate genes, which might be responsible for maintenance of epithelial phenotype. GFP-positive epithelial cells were identified in the original cells, which enable us to identify early EMT markers. By replacing the vimentin promoter with E-cadherin promoter, we could successfully establish a reporter system for mesenchymal-epithelial transition (MET). Through these attempts, we are aiming to identify molecular targets in breast cancer stem cell and triple-negative breast cancer.

Citation Format: Kiyotsugu Yoshikawa, Yoshiaki Matsumoto, Junji Ito, Yoshie Shiojiri, Ellis Reinherz, Masakazu Toi. Application of sensitive EMT reporter to identification of breast cancer stem cell-related genes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C84.
Abstract C9: Human melanoma metastasis in NSG mice correlates with clinical outcome in patients
Authors: Piskounova Elena , Quintana Elsa , Shackleton Mark , Johnson Timothy M. , Morrison Sean J.
Published: August 2014

Although metastasis is the leading cause of cancer-related deaths, little is known about the molecular mechanisms that enable tumor cells to metastasize. We have recently developed a model in which for the first time human melanomas readily engraft in nonobese diabetic/severe combined immunodeficient interleukin-2 receptor-g chain null (NSG) mice. Melanomas from 25 patients exhibit reproducible differences in the rate of spontaneous metastasis after transplantation into NSG mice. Importantly metastasis in mice is strongly correlated with metastasis, and clinical outcome, in the patients, providing an opportunity to test the mechanisms of metastatic progression of human melanoma in an animal model. In transplanted mice, efficiently metastasizing melanomas have much higher levels of circulating melanoma cells compared to inefficiently metastasizing ones, suggesting that the rate of entry into the blood is one factor that limits the rate of metastasis. In addition, we have profiled both genomic mutations and gene expression in 33 primary human melanomas with varying metastatic potential, in order to gain insight into the key regulatory pathways involved in melanoma metastasis. Using our xenograft model we are currently investigating the role of select genes in enhancing melanoma metastasis, at the level of intravasation into the blood stream, survival in circulation and proliferation in distal organs.

Citation Format: Elena Piskounova, Elsa Quintana, Mark Shackleton, Timothy M. Johnson, Sean J. Morrison. Human melanoma metastasis in NSG mice correlates with clinical outcome in patients. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C9.
Abstract IA20: Deregulation of stem cell self-renewal pathways in cancer
Authors: Clarke Michael
Published: August 2014

Most common cancers, such as cancers of the breast and colon, arise in organs such as the breast that contain a small population of stem cells that constantly replenish the mature cells of the tissue. Stem cells are defined by the ability to divide and give rise to a new stem cell (self-renewal), as well as the ability to give rise to the differentiated cells of an organ, and thus are the only long-lived cell population in many tissues. Extensive molecular and cellular analyses factors that play a role in the self-renewal of normal and malignant epithelial stem cells will be discussed.

Citation Format: Michael Clarke. Deregulation of stem cell self-renewal pathways in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA20.
Abstract PR2: Identification of luminal breast cancers that establish a tumor supportive macroenvironment defined by proangiogenic platelets and bone marrow derived cells
Authors: Marsh Timothy , Kuznetsov Hanna , Markens Beth , Castano Zafira , Greene-Colozzi April , Hay Samantha , Brown Victoria , Richardson Andrea , Signoretti Sabina , Battinelli Elisabeth , McAllister Sandra
Published: August 2014

Breast cancer recurrence rates vary following treatment, suggesting that tumor cells disseminate early from primary sites but remain indolent indefinitely before progressing to symptomatic disease. The reasons why some indolent disseminated tumors erupt into overt disease are unknown. We discovered a novel process by which certain luminal breast cancer cells and patient tumor specimens (LBC “instigators”) establish a systemic macroenvironment that supports outgrowth of otherwise-indolent disseminated tumors (“responders”). Instigating LBCs secrete cytokines that are absorbed by platelets, which are recruited to responding tumor sites where they aid vessel formation. Instigator-activated bone marrow cells (BMCs) enrich responding tumor cell expression of CD24, an adhesion molecule for platelets, and provide a source of VEGFR2+ tumor vessel cells. This cascade results in growth of responder adenocarcinomas and is abolished when platelet activation is inhibited by aspirin. These findings highlight the macroenvironment as an important component of disease progression that can be exploited therapeutically.

This abstract is also presented as Poster B6.

Citation Format: Timothy Marsh, Hanna Kuznetsov, Beth Markens, Zafira Castano, April Greene-Colozzi, Samantha Hay, Victoria Brown, Andrea Richardson, Sabina Signoretti, Elisabeth Battinelli, Sandra McAllister. Identification of luminal breast cancers that establish a tumor supportive macroenvironment defined by proangiogenic platelets and bone marrow derived cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr PR2.
Abstract A34: Splenectomy inhibits tumor development and metastases in murine lung cancer models.
Authors: Levy Liran , Mishalian Inbal , Zolotarov Lida , Bayuh Rachel , Fridlender Zvi Gregorio
Published: August 2014

Introduction & Aims: It has been shown that inhibitors of the immune system (e.g. myeloid derived suppressor cells, MDSC) reside in the spleen and inhibit the endogenous anti-tumor effects of the immune system. We hypothesized that excision of the spleen (splenectomy) can inhibit growth of relatively big tumors, and reduce metastases by modulating systemic inhibition of the immune system. Our long-term goal is to implement mechanisms elucidated in these studies into future clinical trials.

Methods: The clinical effect of splenectomy was evaluated in several murine lung cancer models - flank and metastatic. We compared immunological properties of blood and tumor after splenectomy or sham operation in tumor-bearing mice, using FACS analysis, RT-PCR and specific depletion studies.

Results: We found that splenectomy reduces tumor growth, can induce their regression, and decreases metastases. These effects disappeared in NOD/SCID mice. No significant changes in cell types were found in the blood. Splenectomy increased the percentage out of total tumor cells of neutrophils (2.4% vs. 4.9%, p=0.012), and macrophages (10.9% vs. 14.4%, p=0.014). The tumor macrophages tended to be less immune-inhibitory (non-M2/M2 macrophages ratio increased from 3.4 to 12.1, p=0.04). We further noted a tendency to increased activation of CD8+ CTL (19.2% vs. 30%, p=0.09). Tumor microenvironment was found to be more pro-inflammatory following splenectomy (e.g. upregulation of MIG, TNF-alpha, and IFN-gamma. Using specific depletion of cells we evaluated the role of each cell in the effect described. These experiments demonstrated the importance of myeloid derived suppressor cells (MDSC) in the effect of splenectomy.

Conclusions: Splenectomy inhibits the development of tumors and metastases in murine models of lung cancer, by changing the amount and characteristics of myeloid cells, and reducing activation of CD8+ T-cells.

Citation Format: Liran Levy, Inbal Mishalian, Lida Zolotarov, Rachel Bayuh, Zvi Gregorio Fridlender. Splenectomy inhibits tumor development and metastases in murine lung cancer models. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A34.
Abstract A65: Toll-like receptor 3 signaling in ovarian cancer.
Authors: Muccioli Maria , Pate Michelle , Benencia Fabian
Published: August 2014

The purpose of this study is to examine the cytokine secretion (in vitro) and leukocyte infiltration profiles (in vivo) in response to Toll-Like Receptor 3 (TLR3) activation in murine epithelial ovarian carcinoma. Ovarian cancer is the most deadly gynecological neoplasm, typically treated with surgery and chemotherapy, and has a less than a 50% 5-year survival rate. Drug resistance develops frequently, and it is necessary to investigate more targeted therapies. Chronic inflammation, which is seen in many cancers, can render an immunosuppressive tumor microenvironment characterized by cytokines that promote leukocyte infiltration, which can potentiate angiogenesis, and subsequent tumor growth and metastasis. Pathways that can generate such inflammation are important to characterize as they may be targeted in the development of novel cancer therapeutics. One regulator of inflammation in some types of epithelial ovarian cancer is TLR3, a protein that senses dsRNA and activates NF-κB, a potent initiator of cytokine secretion. Although typically expressed in immune cells, TLR3 is also present in cancer cells, and may function to direct inflammation at the tumor site towards an immunosuppressive state capable of recruiting pro-angiogenic leukocytes. Using PCR, ELISA, western blot, and microarray experiments on siRNA-generated TLR3-knock-downs and control samples, we are characterizing the cytokine secretion profiles following TLR3 activation with synthetic dsRNA ligands. Furthermore, we will employ this ovarian cancer cell line (ID8) with C57BL/6 mice to generate transplantation tumors and determine the effects of TLR3 signaling on the leukocyte population and presence of angiogenic molecules at the tumor site using flow cytometry and immunohistochemistry. We have determined that treatment of ID8 epithelial ovarian carcinoma cells with TLR3 ligands poly (I:C) or poly (A:U) results in increased secretion of pleiotropic cytokines, such as CCL5 and IL6, that may potentiate leukocyte migration to the tumor site. In vivo studies with C57BL/6 mice will determine whether this TLR3-induced cytokine secretion in the tumor cells translates to the physiological environment and promotes leukocyte infiltration and angiogenesis. By elucidating the contribution of TLR3 to the recruitment of pro-angiogenic leukocytes in ovarian epithelial carcinoma, the results of this project may be useful in the development of anti-angiogenic therapies for certain ovarian tumors.

Citation Format: Maria Muccioli, Michelle Pate, Fabian Benencia. Toll-like receptor 3 signaling in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A65.
Abstract A67: Reconditioning the tumor immune microenvironment for a breast cancer vaccine therapy.
Authors: Huang Yuhui , Yuan Jianping , Righi Elda , Duda Dan G. , Fukumura Dai , Poznansky Mark C. , Jain Rakesh K.
Published: August 2014

The recent approval of a prostate cancer vaccine has re-energized the research on cancer immunotherapy. A critical barrier for immunotherapies remains the immunosuppressive tumor microenvironment, which is due to abnormal tumor vasculature, hypoxia and tumor-infiltrating myeloid suppressors. Judiciously used anti-angiogenic agents have the potential to modulate the tumor immune environment to improve immunotherapy, but they are often used at high doses in the clinic aiming to prune tumor vessels. However, this has been shown to compromise other concurrent therapies, e.g., chemotherapy. Here, we demonstrate that anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody treatment at lower “vascular normalizing” doses – but not high “anti-vascular” doses – results in a more homogeneous distribution of functional tumor vessels and reduces hypoxia. As a result, the lower doses are superior to the high doses at polarizing tumor-associated macrophages from immune inhibitory M2-like towards immune stimulatory M1-like phenotype, and in facilitating CD4+ and CD8+ T cell tumor infiltration. These findings indicate that vascular normalizing doses of anti-VEGFR2 antibody treatment can reprogram the tumor immune microenvironment from suppressive to stimulatory. Based on this mechanism, synchronizing vascular normalization with T cell activation induced by a whole cancer cell vaccine therapy enhanced anti-cancer efficacy in a CD8+ T cell-dependent manner in both immune tolerant and immunogenic murine breast cancer models. Given that the combinations of high doses of bevacizumab with chemotherapy have not improved overall survival of breast cancer patients, our study suggests a new strategy to more effectively use anti-angiogenic agents with active immunotherapy and potentially other anti-cancer therapies in a clinical setting.

Citation Format: Yuhui Huang, Jianping Yuan, Elda Righi, Dan G. Duda, Dai Fukumura, Mark C. Poznansky, Rakesh K. Jain. Reconditioning the tumor immune microenvironment for a breast cancer vaccine therapy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A67.
Abstract A80: Role of myeloid SOCS3 in prostate cancer progression.
Authors: Yu Hao , Liu Yudong , Benveniste Etty N. , Qin Hongwei
Published: August 2014

Suppressor Of Cytokine Signaling (SOCS) proteins, negative regulators of the JAK/STAT pathway, have been implicated in having a key protective role in tumor initiation and progression. However, the role of SOCS3 in regulating the differentiation and functions of myeloid-derived suppressor cells (MDSC) is poorly understood. Recent studies show the important role of MDSCs in promoting tumor progression as well as metastasis by regulating immune surveillance. To determine the role of myeloid SOCS3 in tumor progression, Conditional SOCS3 deletion in myeloid cells (LysMCre-SOCS3fl/fl) and SOCS3fl/fl C57BL/6 mice were used as recipients for subcutaneous transplantation of TRAMP-derived mouse prostate tumor cells. Our results indicate that tumor growth is significantly enhanced in myeloid-specific SOCS3-deficient mice, which correlates with elevated levels of Gr1+/CD11b+ cells in both spleen and tumor, and less CD8+ T-cell infiltration in tumors. Tumors from myeloid-specific SOCS3-deficient mice also express high levels of Arginase-1 and have elevated Akt signaling. In vitro, SOCS3-deficient bone-marrow-derived mononuclear cells (BDMC) exhibit heightened STAT3 activation and are subject to differentiation towards the MDSC phenotype. These findings suggest that myeloid cell SOCS3 provides protection from tumor progression by regulating MDSC differentiation.

Citation Format: Hao Yu, Yudong Liu, Etty N. Benveniste, Hongwei Qin. Role of myeloid SOCS3 in prostate cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A80.
Abstract A83: Modifications of the bone marrow microenvironment in the transition from monoclonal gammopathy of undetermined significance to multiple myeloma in Vk*MYC mice.
Authors: Calcinotto Arianna , Grioni Matteo , Bertilaccio Maria Teresa Sabrina , Chesi Marta , Bergsagel Leif , Casorati Giulia , Dellabona Paolo , Bellone Matteo
Published: August 2014

Multiple myeloma (MM) is a neoplastic plasma cell (PC) disorder of the oldster, and it is characterized by clonal proliferation of PC in the bone marrow (BM), monoclonal protein in biological fluids and organ dysfunction. While monoclonal gammopathy of undetermined significance (MGUS) often anticipates MM, the role of the cellular components of the BM microenvironment in the shift from MGUS to MM is unknown. Our aim was to define if the immune system has a role in this process. Thus, clinical signs of disease development and progression were investigated in the peripheral blood, bone marrow, kidney and bone of aging Vk*MYC mice to pinpoint the shift from MGUS to MM. We have also defined by immunohistochemistry and multiparametric flow-cytometry the phenotypic and functional characteristics of tumor infiltrating immune cells in the different phases of disease.

We have found that the BM of Vk*MYC mice is the primary organ of PC accumulation, and accrual of PC in the BM associates with the presence of a measurable M-spike. Based on the frequency of PC in the BM and the quantification of M-spike in serum we were able to segregate MGUS from MM. The transition from MGUS to MM in Vk*MYC mice was characterized by increased vascularization of the BM that associated with local enrichment of proangiogenic molecules such as VEGF-A and IL18. The BM was also enriched in molecules that favor survival and proliferation of myeloma cells, and signs of osteoporosis accompanied these alterations. A comparison between Vk*MYC mice affected by MGUS and age matched wild type C57BL/6 mice revealed precocious alteration of the CD4/CD8 T cell ratio, increased percentage of IL2 and IL4 producing CD4+ T cells, and CD8+ T cells more prone to produce IFNγ in the BM of the former.

In summary, our data suggest that early in disease development, yet unknown factors elicit an inflammatory reaction that significantly modifies the cellular composition of the BM and associates with neoangiogenesis and PC accumulation. Being the pathology in the Vk*MYC mouse similar to the human disease, our data might be relevant to understand the mechanisms of progression from MGUS to MM in humans.

Citation Format: Arianna Calcinotto, Matteo Grioni, Maria Teresa Sabrina Bertilaccio, Marta Chesi, Leif Bergsagel, Giulia Casorati, Paolo Dellabona, Matteo Bellone. Modifications of the bone marrow microenvironment in the transition from monoclonal gammopathy of undetermined significance to multiple myeloma in Vk*MYC mice. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A83.
Abstract A92: Tumor suppressive function of human macrophage-derived ADAM15 exosomes.
Authors: Lee Hee Doo , Kim Yeon Hyang , Kim Doo-Sik
Published: August 2014

A disintegrin and metalloproteinase (ADAM) 15, the only ADAM protein containing an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain, is a widely expressed, human membrane protein. The present study demonstrates that macrophages derived from monocytes actively release ADAM15 into the extracellular space as an exosomal component. The exosomal ADAM15 release is further elevated by lipopolysaccharide stimulation, indicating that the exosome release is closely associated with immune cell differentiation and activation. ADAM15-rich exosomes display enhanced binding affinity for integrin alpha v beta 3, thereby interfering with the interaction between integrin alpha v beta 3 and vitronectin. ADAM15 exosomes significantly suppress vitronectin- and fibronectin-induced proliferation and migration of various tumor cells as well as tumor growth in vivo. Tumor suppressive function of exosomal ADAM15 is effectively blocked by monoclonal antibody specific for the extracellular domain of ADAM15. Experimental evidence shows that RGD-containing disintegrin-like domain, but not metalloproteinase activity, of ADAM15 is the responsible element for the tumor suppressive activity. Notably, proliferation and migration of macrophages are stimulated by the exosomes, indicating anti-tumor immunity. This work strongly suggests that a novel tumor suppressive mechanism is mediated by macrophage-derived ADAM15 exosomes.

Citation Format: Hee Doo Lee, Yeon Hyang Kim, Doo-Sik Kim. Tumor suppressive function of human macrophage-derived ADAM15 exosomes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A92.
Abstract B23: DNA damage response in responder lymphocytes reduces vaccine efficacy following alkylating chemotherapy.
Authors: Litterman Adam J. , Zellmer David M. , Grinnen Karen L. , Dudek Arkadiusz Z. , Largaespada David A. , Ohlfest John R.
Published: August 2014

The effect of alkylating chemotherapy on cancer vaccines is controversial, with numerous reports of immune stimulation via Treg depletion following cyclophosphamide and temozolomide treatment. Using a pre-clinical model of a cancer vaccine in both naïve and tumor bearing animals, we observed that the direct anti-proliferative effect of drugs such as temozolomide or cyclophosphamide reduced the quantity of vaccine induced immune responses in a dose dependent manner. Amounts of temozolomide exposure in mice that duplicated human pharmacokinetic exposure and lymphopenia led to lower numbers of CD8 T cells elicited by vaccination with the model antigen ovalbumin. In vitro experiments with purified OT-I CD8 T cells implicated activation of DNA damage responses within strongly proliferating cells as the cause of this diminished immunity. ATM phosphorylation was greater in temozolomide or cyclophosphamide exposed OT-I cells stimulated with the canonical SIINFEKL peptide relative to variant peptides that trigger weaker TCR signaling. In vaccinated mice, this activation of DNA damage response in lymphocytes receiving the highest intensity TCR signals lead to lower avidity of responder cells for cognate antigen and lower effector function. These quantitative and qualitative defects of vaccine driven responses lead to inferior efficacy and loss of survival benefit in murine models. Mice bearing ovalbumin peptide expressing glioma had faster tumor growth and shorter survival following temozolomide and vaccine relative to vaccine only treated mice. Similar defects of vaccine generated responses and a loss of survival benefit relative to controls were observed in response to a neo-antigen vaccine following cyclophosphamide pre-treatment in melanoma bearing mice. The clinical relevance of this finding is demonstrated by a phase II cancer vaccine trial in metastatic melanoma patients where vaccine only patients survived a median of 4.2 years versus less than 8 months in patients treated with low dose cyclophosphamide before vaccine.

Citation Format: Adam J. Litterman, David M. Zellmer, Karen L. Grinnen, Arkadiusz Z. Dudek, David A. Largaespada, John R. Ohlfest. DNA damage response in responder lymphocytes reduces vaccine efficacy following alkylating chemotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B23.
Abstract B3: CD39+ cancer cells mediate immunosuppression reverted by CD39-blocking antibodies.
Authors: Regairaz Anne , Laheurte Caroline , Bonnefoy Nathalie , Eliaou Jean-Francois , Alberici Gilles , Bensussan Armand , Bastid Jeremy
Published: August 2014

The CD39 and CD73 ectonucleotidases hydrolyze extracellular ATP and ADP into immunosuppressive adenosine that binds adenosine receptor and inhibits T cell and NK cell responses. It has been demonstrated that CD39+ Tregs are increased in some human cancers and participate to immunosuppression. The importance of CD39+ Tregs in promoting tumor growth and metastasis has been evidenced in several models in vivo (for a review, see Bastid J et al., Oncogene, 2012). Here, we addressed whether CD39 is expressed by tumor cells and whether CD39+ tumor cells mediate suppression through the CD39-adenosine pathway. Immunohistochemical staining of normal and tumor tissues revealed that CD39 is upregulated in several types of human cancer. In cancer specimens, CD39 is expressed by infiltrating lymphocytes, tumor stroma and tumor cells, whereas its expression in normal samples is absent or weak and mostly limited to vascular endothelia. The expression of CD39 at the cell surface of tumor cells was further directly demonstrated by flow cytometry in human cancer cell lines. We evidenced that CD39+ tumor cells express functional CD39 and inhibit CD4 and CD8 T cell function in a CD39-dependant manner. Treatment with CD39-blocking antibodies was able to alleviate CD39+ tumor cell-mediated inhibition of CD4 and CD8 T cells in co-culture experiments. In conclusion, interfering with the CD39-adenosine pathway could represent a novel immunotherapy strategy for inhibiting Treg and tumor cell-mediated immunosuppression. Furthermore, these results support the ongoing development of CD39-blocking monoclonal antibodies as potential anticancer drugs.

Citation Format: Anne Regairaz, Caroline Laheurte, Nathalie Bonnefoy, Jean-Francois Eliaou, Gilles Alberici, Armand Bensussan, Jeremy Bastid. CD39+ cancer cells mediate immunosuppression reverted by CD39-blocking antibodies. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B3.
Abstract B37: The heat shock protein receptor CD91 is necessary for tumor antigen cross-priming in vivo.
Authors: Zhou Yu Jerry , Binder Robert J.
Published: August 2014

Immunosurveillance mechanisms of tumors depend on the host's ability to cross-prime minute quantities of tumor antigens. We have investigated the role of tumor-derived heat shock proteins (HSPs) in transfer of tumor antigens from tumor cells to antigen presenting cells for two reasons; 1) HSPs are known to chaperone peptides in the cells. The chaperoned peptides reflect the entire antigenicity of the cell from which they are derived. 2) HSPs activate antigen presenting cells resulting in the provision of co-stimulatory molecules and cytokines. These two events require the cell surface receptor CD91 that is expressed on APCs. Here we have used a combination of CD91 inhibitors and CD91 knock-out mice to demonstrate that the HSP-CD91 interaction is crucial for developing concomitant immunity routinely seen in the tumor bearing host. This pathway is indispensable for cross-presentation when antigen levels are low. Considering that elevated levels of endogenous CD91 inhibitors have been reported in colon cancer patients, we propose a novel mechanism of immune evasion for tumors expressing competing ligands for immune receptors such as CD91.

Citation Format: Yu Jerry Zhou, Robert J. Binder. The heat shock protein receptor CD91 is necessary for tumor antigen cross-priming in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B37.
Abstract B39: Activated B cells infiltrate lung cancer.
Authors: Bruno Tullia C. , Kern Jeffrey , Slansky Jill E.
Published: August 2014

Lung cancer is the leading cause of cancer death in men and women, both in the United States and the world. The current 5-year survival rate for lung cancer in the United States is 16%, which can be attributed to the fact that the majority of lung cancer subjects present with late-stage disease that is not curable by current therapies i.e. chemotherapy and radiation. However, in the last several years, novel therapies have emerged to make lung cancer therapy better tolerated and more effective. We are particularly interested in developing an immunotherapy that will help to engage the immune system in targeting and destroying tumor cells. In a comprehensive survey of the immune infiltrate in non-small cell lung cancer (NSCLC) patients, we found that the frequency and total numbers of B cells (TIL-Bs) in the tumor versus the tumor-adjacent tissue was increased in comparison to other immune subsets, specifically, CD4, CD8, and regulatory T cells as well as NK and NKT cells. Further, these TIL-Bs were activated (CD21lo and CD69+), proliferated, and acquired a memory phenotype (CD27+). To test the function of the TIL-Bs, we are determining the types of cytokines they produce, their ability to secrete total IgG, and their potential to present antigen to CD4 T cells. Preliminary studies indicate that these cells generate activation cytokines (IFN-γ and IL-12) rather than suppressive cytokines, which suggests that the TIL-Bs have an effector phenotype that could aid in targeting the tumor. In addition, we are exploring why the TIL-Bs migrate to the site of the tumor. Preliminary data suggest that the common B cell chemokine receptors (CXCR3, CXCR4, CXCR5, CXCR7) are not elevated in TIL-Bs in the tumor compared to tumor adjacent tissue. However, normal peripheral blood B cells migrate toward tumor cell supernatant compared to tumor adjacent cell supernatant, which leads to the hypothesis that the tumor microenvironment generates more chemokines that attract TIL-Bs to the site of the tumor. Ultimately, we aim to identify a novel target for an immunotherapy clinical trial in lung cancer patients.

Citation Format: Tullia C. Bruno, Jeffrey Kern, Jill E. Slansky. Activated B cells infiltrate lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B39.