bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3446: Fibroblast growth factor 23 promotes prostate cancer cell proliferation and invasiveness in vitro
Authors: Feng Shu , Ittmann Michael
Published: May 2015

Prostate cancer is the most common malignancy among American men and remains the second leading cause of male cancer mortality. Since age is thought to be one of the crucial factors in prostate cancer development, fibroblast growth factor 23 (FGF23), which forms a complex with Klotho, the age-related gene product, could play a key role in prostate development and progression. FGF23 is a peptide hormone member of the endocrine FGF subfamily (FGF19, FGF21, and FGF23), which signals through FGF receptors (FGFRs) in the presence of the coreceptor α-Klotho. FGF-23 has a critical role in phosphate homeostasis and vitamin D metabolism. The regulation of 1,25-Dihydroxyvitamin D by FGF-23 could also have implications for cancers, given that vitamin D metabolites have been shown to have anticarcinogenic effects. It has been reported that higher concentrations of FGF-23 were found in patients with advanced ovarian cancer compared with those with early-stage cancer or control subjects; circulating FGF-23 is associated with an increased risk of colorectal adenoma; however, to date, no investigations of the role of FGF-23 in prostate cancer have been reported. In the present study we investigated the function of FGF23 on human prostate cancer progression. FGF23 gene expression was determined in the commonly used prostate cancer cell lines: PC3, LNCaP, DU145, VCaP, and an immortalized normal prostate cell line PNTIA. RT-PCR showed that both FGF23 and αKlotho (KLA) genes are expressed in all these cell lines. Quantitative real-time RT-PCR indicated that FGF23 gene expression is elevated in prostate cancer compared to normal prostate tissue; FGF23 expression frequency is significantly higher in prostate cancer in compare with normal tissue (prostate cancer: total case number 35, positive 23; normal prostate tissue: total number 33, positive 15; p=0.04841). Biological function assays demonstrated that exogenous FGF23 stimulates prostate cancer cell proliferation (LNCaP by 30%, PC3 20%), anchorage-independent growth (by 60% for both LNCaP and PC3 cells), and invasion (LNCaP by 50% and PC3 55%). Suppression of FGF23 expression by FGF23 short hairpin RNA (shRNA) significantly inhibits PC3 and LNCaP cell proliferation, soft agar colony formation, and invasiveness in vitro. These data suggest that FGF23 promotes human prostate cancer progression and targeting FGF23 signaling pathway could decrease prostate cancer growth and metastasis.

Citation Format: Shu Feng, Michael Ittmann. Fibroblast growth factor 23 promotes prostate cancer cell proliferation and invasiveness in vitro. [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 3446. doi:10.1158/1538-7445.AM2014-3446
Abstract 3458: Role of the EphB1 gene in mediating migration, proliferation, and radiosensitization of medulloblastoma cells
Authors: Baig* Nimrah A. , Timofeeva* Olga , Dritschilo Anatoly , Pasquale Elena , Kool Marcel , Rood Brian , Rodriguez Olga , Albanese^ Christopher , Karam^ Sana
Published: May 2015

Eph/ephrin signaling in a variety of cancers has been reported to promote aspects of tumorigenesis, including proliferation, migration, and angiogenesis. Medulloblastoma is an aggressive primitive neuroectodermal tumor originating in the cerebellum. Recently performed integrative genomic approach to a large cohort of medulloblastomas has identified four subtypes (A-D) based on clinical presentation, transcription profiles, genetic abnormalities, and clinical outcome. We show high levels of EphB1 expression in tumors derived from children with group D medulloblastomas, which are known to exhibit a poorer response to current therapies. The purpose of this study is to examine the effect knockdown of EphB1 has on cellular migration, proliferation, and radiosensitization of medulloblastoma tumors. Using a human-derived medulloblastoma cell line (DAOY) and small interfering RNA (siRNA) targeting EphB1, we compared cell migration in an electrical impedance-based transwell Boyden chamber assay (xCELLigence RTCA DP system). We show that siRNA knockdown of EphB1 results in significantly decreased migration. Assessment of proliferation by MTT assay indicates that knockdown of EphB1 affects cell cycle progression in DAOY. Results from cell cycle and sub-G1 analysis by flow cytometry demonstrate that knockdown of EphB1 results in G1 arrest. Our previous systems analysis investigation of global gene expression regulated by an intact ATM gene product also identified EphB1 as a downstream signaling molecule correlating with enhanced cellular radiation resistance. Our data strongly support EphB1 as an unequivocal target for radiation sensitization in human fibroblasts as well as in vitro medulloblastoma cell lines. siRNA knockdown of EphB1 expression in human medulloblastoma cell lines known to express high levels of EphB1 results in sensitization to ionizing radiation by clonogenic survival assays. Preliminary data from an in vivo xenograft medulloblastoma animal model suggest an enhanced response to radiation in tumors lacking the EphB1 gene. Our data indicate that EphB1 is a potential molecular target for medulloblastoma cancer treatment and radiosensitization. The mechanisms and downstream signaling cascades underlying EphB1's action on cell migration, proliferation, and cellular radiosensitization are currently under investigation.

Citation Format: Nimrah A. Baig*, Olga Timofeeva*, Anatoly Dritschilo, Elena Pasquale, Marcel Kool, Brian Rood, Olga Rodriguez, Christopher Albanese^, Sana Karam^. Role of the EphB1 gene in mediating migration, proliferation, and radiosensitization of medulloblastoma 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 3458. doi:10.1158/1538-7445.AM2014-3458
Abstract 3493: An in-situ proximity assay using biotin ligase
Authors: Jiang Zeyu (David) , Hong Rui , Farrell Mike
Published: May 2015

Protein-protein interactions (PPIs) are fundamental to biological processes, from signaling pathways that modulate gene expression, cell proliferation, tumor invasion to cytoskeleton networks that maintain morphological integrity of cells and tissues. Activation of oncogenes and inactivation of tumor suppressor genes in the cell that lead to carcinogenesis are inevitably accompanied by disruption of normal or activation of aberrant PPIs, thus detection and understanding of PPIs may advance the diagnosis and treatment of cancer.

Although many methods have been developed to discover and detect PPIs in vivo and ex vivo, few are applicable to in situ detection of PPIs, particularly in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Here we report a sensitive in situ PPI detection method for FFPE tissues that utilizes biotin ligase (BirA), an enzyme that covalently labels its specific peptide target substrate (BTS) with biotin. We developed BirA and BTS conjugated antibodies, each of which binds specifically to one of the interacting protein pair and whose close proximity permits BirA to biotinylate the BTS for subsequent visualization.

Using both artificial system such as hapten-labeled proteins and known biological PPI models such as E-cadherin and beta-catenin and HER2/HER3 dimers in the context of FFPE tissues, we have demonstrated that the assay is both sensitive and robust. Moreover, the assay has been fully automated and is easily adaptable for detecting protein dimers, fusion proteins, or other novel binary interactions in tissue specimens. As understanding details of signaling pathways becomes increasingly important in cancer biology, in situ proximity assay may offer novel mechanistic and therapeutic insights and provide a powerful tool for cancer diagnostics.

Citation Format: Zeyu (David) Jiang, Rui Hong, Mike Farrell. An in-situ proximity assay using biotin ligase. [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 3493. doi:10.1158/1538-7445.AM2014-3493
Abstract 3519: The c-Myc oncogene over-activates dormant replication origins and sensitizes cancer cells to chemotherapy drugs
Authors: Bryant Victoria , Alexandrow Mark G.
Published: May 2015

Several clinical studies have shown a correlation between elevated c-Myc levels and a better response to chemotherapy. Although elevated c-Myc might indirectly cause such chemosensitivity, it is possible that a direct effect is occurring due to underlying molecular mechanisms. The purpose of the current study was to determine whether c-Myc overexpression could acutely cause chemosensitivity. The c-Myc protein is known to activate DNA replication origins, but the mechanism for this is unknown. Origins are regulated by the loading of MCM2-7 hexamers, which form the core of the helicase that starts the initiation of replication. Mammalian cells load more MCM complexes than are necessary to complete one round of replication. The excess MCM complexes function as backups to allow recovery of replication when forks stall during drug treatment. Consistent with this, we found that loss of Mcm4 or Mcm7 sensitizes PDAC cell lines to 5-FU and Gemcitabine, two S phase stress drugs that are used to treat PDAC patients but are not highly effective on their own. Similar to depletion of backup MCM complexes via loss of Mcm4 or Mcm7, elevated c-Myc expression over-activates backup MCMs, leaving fewer available to recover from drug treatment. Cells containing overexpressed c-Myc display increased chemosensitivity to Gemcitabine that is not seen in cells containing normal c-Myc levels or a non-functional c-Myc mutant. These results demonstrate that reduction in backup MCM complexes, either by direct downregulation or by oncogene driven over-activation (via c-Myc), render cells sensitive to chemotherapy drugs. These results suggest that development of drugs that target MCM functionality may be useful in combination with S-phase stress drugs currently used in the clinic. These results also demonstrate that c-Myc has the ability to acutely cause chemosensitivity and c-Myc elevated tumors may respond better to chemotherapy due to the c-Myc effects on MCM complexes.

Citation Format: Victoria Bryant, Mark G. Alexandrow. The c-Myc oncogene over-activates dormant replication origins and sensitizes cancer cells to chemotherapy drugs. [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 3519. doi:10.1158/1538-7445.AM2014-3519
Abstract 352: Integrating transcriptomic data using metacore pathway analysis to identify novel biomarkers of bevacizumab target engagement
Authors: Willis Chris D. , Lafferty-Whyte Kyle , Baker Melinda , Pestell Richard G.
Published: May 2015

The widespread use of microarrays for gene expression studies has generated large volumes of transcriptomic data to help describe mechanisms such as drug treatment response. The drug Bevacizumab is well characterized for its ability to inhibit VEGFA pro-angiogenic signaling resulting in tumor regression for a number of cancer-related indications. However, universal mechanisms of action have remained elusive to describe changes at the mRNA level across different solid tumor types. This case study uses 3 publically available datasets related to Bevacizumab treatment and analyzes the data in a manually curated systems biology context. Using a combination of Thomson Reuters solutions including the Data Annotation and Processing tool, MetaCore Pathway Analysis and Integrity Biomarkers Module allows for a simple workflow to process omics data and identify biomarkers of target engagement through pathway analysis and data integration. This analysis identified 5 differentially expressed genes common to all treated tumors including: Basonuclin, growth arrest-specific 7, mucin 1, integrin beta-4, and dystonin. All 5 of these objects can be connected to VEGF-A in a signaling network based on previously curated interactions. These in silico results provide a strong rationale for follow up studies in the lab to validate novel biomarkers related to Bevacizumab target engagement in solid tumors.

Citation Format: Chris D. Willis, Kyle Lafferty-Whyte, Melinda Baker, Richard G. Pestell. Integrating transcriptomic data using metacore pathway analysis to identify novel biomarkers of bevacizumab target engagement. [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 352. doi:10.1158/1538-7445.AM2014-352
Abstract 3547: FoxD3-regulated microRNA-137 suppresses tumour growth and metastasis in human hepatocellular carcinoma by targeting AKT2
Authors: Liu Li-Li , Lu Shi-Xun , Li Min , Fu Jia , Zhang Chris Zhiyi , Yun Jing-Ping
Published: May 2015

Background and Aims: microRNAs, which are frequently deregulated in human cancer, have been implicated in the progression of hepatocarcinogenesis. Our study aims to investigate the role of miR-137 in cell proliferation and metastasis of hepatocellular carcinoma and the underlying molecular mechanisms.

Methods: Real-time PCR and western blot were used to examine the expressions of miR-137, Akt2 and FoxD3 in HCC tissues and cells. The in vitro and in vivo effects of miR-137 were determined. Luciferase reporter assay was conducted to confirm the association of miR-137 and AKT2. CHIP assay was performed to test the regulation of miR-137 by FoxD3.

Results: We show that microRNA (miR)-137 is significantly down-regulated in hepatocellular carcinoma (HCC), and its decreased expression is associated with vein invasion, incomplete Involucrum, and tumour metastasis. Multivariate analysis suggests that miR-137 is an independent indicator for poor survival. To determine its role in HCC development, we showed that overexpression of miR-137 suppresses cell proliferation, invasion and metastasis in vitro. Conversely, miR-137 inhibition promoted HCC cell growth. We also identified AKT2 as a key target of miR-137 in this context. Statistical data revealed a reverse correlation of AKT2 and miR-137 expression in HCC patients. Silencing of AKT2 phenotypically copied miR-137-induced phenotypes, whereas re-expression of AKT2 reversed the suppressive effects of miR-137. Further investigation showed that miR-137 exerted its anti-tumour activity via inhibiting the AKT2/mTOR pathway. Moreover, we demonstrated that FoxD3 binds to the promoter of miR-137 and activates its transcription. In vivo studies confirmed that FoxD3-regulated miR-137 inhibition of HCC growth and metastasis via directly targeting AKT2.

Conclusions: Our findings indicated that miR-137 is a valuable biomarker for HCC prognosis and demonstrated that the FoxD3/miR-137/AKT2 regulatory network plays an important role in HCC progression.

Citation Format: Li-Li Liu, Shi-Xun Lu, Min Li, Jia Fu, Chris Zhiyi Zhang, Jing-Ping Yun. FoxD3-regulated microRNA-137 suppresses tumour growth and metastasis in human hepatocellular carcinoma by targeting AKT2. [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 3547. doi:10.1158/1538-7445.AM2014-3547
Abstract 3548: Study of the tumor suppressive machinery of arsenic trioxide-induced glioblastoma multiforme inhibition via microRNA-182-Sestrin2 regulatory circuit
Authors: Lin Liang-Ting , Chiou Shih-Hwa , Lee Yi-Jang
Published: May 2015

Glioblastoma multiforme (GBM) is classified the fourth grade of human glioma that indicates the most malignant brain tumor with poor five-year survival (5%). Accumulated evidence showed that GBM cells were resistant to various therapeutic strategies. Recently, use of arsenic trioxide (ATO) to induce apoptosis and to increase the radiosensitivity was reported for the benefit in treating GBMs. However, the responsible signaling cascades are still remain investigation. MicroRNA, a category of RNA interference, regulates its downstream genes post transcriptionally by binding to the 3′untraslated region (UTR) of its targets. In this study, we used S1 and S1R1 GBM cells which were derived from the primary and the recurrent lesion respectively. Firstly, we identified that an oncogenic microRNA, miR-182, was down-regulated after the treatment of ATO in both GBM cells. In addition, one of the miR-182 targeted coding genes, Sestrin 2 (SESN2), plays an important role in relieving intracellular oxidative stress and participates in TP53 tumor suppressive pathway. Use of luciferase-based reporter assay confirmed that miR-182 directly bound to SESN2 3′UTR and interrupted the further translation. Intriguingly, the basal level of miR-182 in the primary S1 GBM was significantly lower than the recurrent S1R1 GBM, supporting the oncogenic evidence in the previous studies. The counteracted SESN2 expression was also determined by real-time qPCR and western blot. We then hypothesized that ATO may retarded the growth of GBM through a miR-182-SESN2 related regulatory axis. Overexpression of miR-182 in S1 GBM resulted in a decrease of SESN2 and insensitive to ATO treatment. On the opposite, inhibition of miR-182 in S1R1 GBM dramatically retarded the growth rate and sensitized the cells to ATO. Moreover, pretreatment of an antioxidant, N-acetyl-cysteine (NAC), rescued the miR-182 suppression, suggesting that ATO-induced oxidative stress may be dominant in this responsive axis. Concordant expression of miR-182/96/182 cassette was revealed, indicating the transcriptional regulation was unveiled. Further investigation on the detailed machinery still remain elucidating. Based on our findings, we displayed that ATO-induced oxidative stress triggered a tumor suppressive response through a miR-182-SESN2 dependent machinery.

Citation Format: Liang-Ting Lin, Shih-Hwa Chiou, Yi-Jang Lee. Study of the tumor suppressive machinery of arsenic trioxide-induced glioblastoma multiforme inhibition via microRNA-182-Sestrin2 regulatory circuit. [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 3548. doi:10.1158/1538-7445.AM2014-3548
Abstract 3554: Gene and miRNA expression networks specific to never smoker lung adenocarcinoma
Authors: Kusko Rebecca , Garrison Carly , Wang Teresa , Campbell Josh , Perez-Rogers Joseph , Luo Lingqi , Beane Jennifer , Liu Gang , Kadara Humam , Belinsky Steven , Lenburg Marc E. , Spira Avrum
Published: May 2015

While smoking is well recognized as a major risk factor for lung cancer, there is a growing incidence of lung cancer in never smokers which is in turn the fifth leading cause of cancer-related death worldwide. Never smokers (NS) who develop lung cancer exhibit disparate profiles of somatic mutations and clinical responses to targeted therapy relative to lung cancer arising in current or former “ever” smokers (ES), suggesting that ES and NS lung cancer arise through distinct molecular processes. We therefore sought to characterize mRNA and miRNA expression differences specific to NS adenocarcinoma (AdC) to gain insights into the molecular differences underlying NS and ES AdC carcinogenesis.

Total RNA was isolated from matched pairs of lung AdC tumor and adjacent histologically normal tissue obtained from 22 subjects (8 NS, 14 ES). Large and small RNA libraries were sequenced on the Illumina HiSeq 2000. Tumor-specific gene and miRNA expression differences between NS and ES were identified using linear mixed-effects ANOVA. MiRConnx was used to construct miRNA-mRNA networks.

We identified 120 mRNA and 15 miRNA whose expression was modified uniquely in NS lung AdC. In the predicted miRNA-mRNA regulatory network, additional analysis pinpointed modulation of the development and cellular metabolism canonical pathway within genes connected to several of the differentially expressed miRNA (GATHER, p<0.001). Several of the differentially expressed mRNA and miRNA were validated by qRT-PCR in samples from an independent cohort.

In summary, the construction of a miRNA-mRNA regulatory network has enabled us to identify molecular alterations that may be specific to NS lung AdC. Ultimately, these findings may serve to broaden the landscape of personalized therapeutic and treatment options by identifying targetable molecular interactions and therapeutic drug candidates for lung AdC in never smokers.

Citation Format: Rebecca Kusko, Carly Garrison, Teresa Wang, Josh Campbell, Joseph Perez-Rogers, Lingqi Luo, Jennifer Beane, Gang Liu, Humam Kadara, Steven Belinsky, Marc E. Lenburg, Avrum Spira. Gene and miRNA expression networks specific to never smoker lung adenocarcinoma. [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 3554. doi:10.1158/1538-7445.AM2014-3554
Abstract 3571: mRNA spike-in control materials for cancer fusion gene detection assays
Authors: Liang Winnie , Pond Stephanie J. , Tembe Waibhav D. , Chuang Han-Yu , Legendre Christophe , Kim Nancy , Montel Valerie , Wong Shukmei , McDaniel Timothy K. , Craig David , Carpten John
Published: May 2015

Oncogenic fusion genes underlie the mechanism of several common cancers and also constitute or encode important diagnostic and therapeutic targets. Studies using next-generation sequencing technologies, particularly RNA sequencing (RNA-seq), have revealed a growing number of recurrent fusions in a variety of cancers. As such, establishing analytic parameters, including the limit of detection, sensitivity, and specificity of detecting fusions in tumor RNA specimens is important. We have developed reference materials useful in assessing the ability to detect fusion events in RNA or DNA samples. These consist of a set of nine synthetic poly-adenylated RNA transcripts that correspond to common cancer fusion gene sequences. These synthetic fusion RNAs can be spiked at known concentrations into total RNA in the first stage of the mRNA sample prep and used as in-line controls to evaluate the sensitivity and specificity of the detection of these mutations. As a demonstration of the utility of these controls, we performed a series of spike-in experiments to investigate performance characteristics of three commonly used fusion-detection software algorithms. Our results show all three algorithms to be capable of detecting fusions in a dose-dependent manner. The results also show differences in performance among the algorithms for detecting specific fusions.

Citation Format: Winnie Liang, Stephanie JK Pond, Waibhav D. Tembe, Han-Yu Chuang, Christophe Legendre, Nancy Kim, Valerie Montel, Shukmei Wong, Timothy K. McDaniel, David Craig, John Carpten. mRNA spike-in control materials for cancer fusion gene detection assays. [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 3571. doi:10.1158/1538-7445.AM2014-3571
Abstract 3585: Metastasis of 4T1 murine breast cancer cells to the lung is dependent on the chemokine CCL2/MCP-1 produced by stromal cells in the primary tumor
Authors: Yoshimura Teizo , Howard O.M. Zack , Ito Toshihiro , Kuwabara Masaki , Matsukawa Akihiro , Chen Keqiang , Liu Ying , Liu Mingyong , Wang Ji Ming
Published: May 2015

The chemokine CCL2, also known as MCP-1, plays an important role in the initiation and progression of cancer. Since tumor cells produce CCL2, they are considered to be the main source of this chemokine. Here, we examined the capacity of CCL2 produced by non-tumor cells to affect the growth and lung metastasis of 4T1 mouse breast cancer cells. Primary tumors formed in the mammary pad of WT or CCL2-/- mice grew at a similar rate; however, lung metastases were markedly reduced in CCL2-/- mice, with significantly longer mouse survival. High levels of CCL2 mRNA were detected in tumors growing in WT, but not CCL2-/- mice. Serum CCL2 levels were increased in tumor-bearing WT, but not CCL2-/- mice. Transplantation of CCL2-/- bone marrow cells into WT mice did not alter the incidence of lung metastasis, but transplantation of WT bone marrow cells into CCL2-/- mice increased lung metastasis. The primary tumors of CCL2-/- mice consistently developed necrosis earlier than those of WT mice and showed decreased infiltration by macrophages and reduced angiogenesis. Interestingly, 4T1 cells that metastasized to the lung constitutively expressed elevated levels of CCL2. Intravenous injection of 4T1 cells producing a high level of CCL2 resulted in increased tumor foci in the lung of CCL2-/- mice. Finally, the culture supernatants of 4T1 cells markedly induced CCL2 production in mouse macrophages. Thus, CCL2 produced by tumor cell-activated stromal cells in the primary tumors promotes lung metastasis of 4T1 cells, while tumor cell-derived CCL2 also contributes to the metastasis once tumor cells enter the circulation. A greater understanding of the source and role of CCL2 during tumor progression and the mechanisms by which stromal cells produce this chemokine may lead to novel strategies for cancer treatment.

Citation Format: Teizo Yoshimura, O.M. Zack Howard, Toshihiro Ito, Masaki Kuwabara, Akihiro Matsukawa, Keqiang Chen, Ying Liu, Mingyong Liu, Ji Ming Wang. Metastasis of 4T1 murine breast cancer cells to the lung is dependent on the chemokine CCL2/MCP-1 produced by stromal cells in the primary tumor. [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 3585. doi:10.1158/1538-7445.AM2014-3585
Abstract 3632: Targeting of tumor myeloid suppressor cells by Salmonella bacteria causes a shift to M1 phenotype and leads to inhibition of tumor growth
Authors: Al-Ramadi Basel K. , Kaimala Suneesh , Mohamed Yassir A. , Issac Jincy M. , Elkord Eyad , Chouaib Salem , Fernandez-Cabezudo Maria J.
Published: May 2015

Tumor development is associated with changes in hematopoiesis that lead to an increase in myelomonocytic cells in systemic sites as well as within the tumor tissue. The accumulation of myeloid cells within tumors not only promotes angiogenesis and tissue remodeling required for tumor growth but also maintains the tumor milieu as an immunosuppressive environment. We have previously shown that attenuated strains of the facultative anaerobic bacteria, Salmonella enterica serovar Typhimrium, can effectively retard tumor growth in a therapeutic melanoma model. Given that Salmonella organisms utilize host macrophages as their primary niche for survival, we investigated the consequences of Salmonella treatment on myeloid cell recruitment, phenotypic characteristics and functional activation within tumor tissue of B16.F1 melanoma-bearing animals. Treatment with Salmonella led a preferential expansion, within spleen and tumor tissues, of CD11b+Gr-1+, but not CD11b+Gr-1-, myeloid cells. Using 6-color FACS analysis, CD11b+Gr-1+ cells were shown to exhibit significant up-regulation in the expression of activation markers, including MHC class II, CD80, and Sca-1/Ly6A proteins. Moreover, the functional activities of total tumor infiltrating cells or CD11b-purified cells from tumor tissue were assessed by qRT-PCR. Salmonella treatment induced a dramatic increase in iNOS and IFN-g gene expression but inhibited ARG1 and IL-4 genes. Further, while expression of TGF-b and VEGF was reduced, the level of inflammation-related S100A9 gene was increased. Our findings demonstrate that systemic administration of attenuated Salmonella leads to phenotypic and functional changes in tumor myeloid suppressor cells, with a shift from a predominantly M2 phenotype to an activated, effector macrophage phenotype.

Citation Format: Basel K. Al-Ramadi, Suneesh Kaimala, Yassir A. Mohamed, Jincy M. Issac, Eyad Elkord, Salem Chouaib, Maria J. Fernandez-Cabezudo. Targeting of tumor myeloid suppressor cells by Salmonella bacteria causes a shift to M1 phenotype and leads to inhibition of tumor growth. [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 3632. doi:10.1158/1538-7445.AM2014-3632
Abstract 3633: Combination of innate immune activators stimulates leukocyte populations in the tumor microenvironment
Authors: Shahar Ehud , Gorodetsky Raphael , Aizenshtein Elina , Pitcovski Jacob
Published: May 2015

Tumor microenvironment (TME) harbors a multitude of immune cells which can comprise up to 50% of the tumor. The most abundant cells in the TME often are tumor associated macrophages (TAM) and tumor associated neutrophils (TAN). These cells potentially possess anti-tumoral cytotoxic effects, and have the potential to induce a broad range of cellular immune responses. However, in the developing tumor, due to elements secreted by the tumor cells, TAM and TAN are altered to act in a pro-tumor manner. The purpose of our study was the optimization and characterization of a treatment aimed at inducing an anti-tumoral response of innate immune cells in the TME. In a melanoma B16 mice model, micro-particles carrying targeting and induction components were injected intra-tumorally and the responses of the tumors, including immune cells populations were monitored. Without treatment the CD45+ cells ratio of total TME decreased as tumor progressed. Particles carrying Toll-like receptor ligands inhibited tumor growth, inducing significant elevation of CD45+ cells in the TME. Using different inducing molecules (inducers) or simultaneous use of several inducers enabled the stimulation of specific immune cell populations within the tumor. The current study suggests that by using tumor targeted particles with adequate inducers, one could change the tumor microenvironment innate immune cells toward an anti-tumor response.

Citation Format: Ehud Shahar, Raphael Gorodetsky, Elina Aizenshtein, Jacob Pitcovski. Combination of innate immune activators stimulates leukocyte populations in the tumor microenvironment. [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 3633. doi:10.1158/1538-7445.AM2014-3633
Abstract 3636: Anti-CD20 therapy prevents protumor regulatory T-cell expansion and triggers a memory Th1 response in tumor-bearing mice
Authors: Deligne Claire , Siberil Sophie , Teillaud Jean-Luc
Published: May 2015

The long-lasting clinical response by lymphoma patients to anti-CD20 treatment has been attributed to a ‘vaccinal’-type effect through the induction of an anti-tumor adaptive immunity. Using a model where C57Bl/6 mice were injected intravenously with EL4 tumor cells expressing human CD20, we previously showed that CD4 cells are central to the host protection induced by anti-CD20 therapy. Here, we have first shown that the transfer of highly purified CD4+ T cells induces a long-term protection, demonstrating that this T-cell subset plays indeed a key role in the induction of an adaptive memory response. In addition, CD4+ effector memory T cells were detected in surviving animals. We have then analyzed the changes occurring in various T cell subsets in tumor-bearing animals receiving anti-CD20 treatment. On the one hand, untreated animals exhibited a large expansion of CD4+FoxP3+ regulatory T cells (Tregs), with reduced survival. Depletion of these cells using anti-CD25 injection led to long-term survival, indicating that a Treg-dependent immuno-suppressive environment is established after tumor injection in this model. On the other hand, anti-CD20 therapy, that induces a long-lasting protection, reversed the initial expansion of Tregs one week after the end of anti-CD20 treatment, and was accompanied by a marked increase in the number of Th1 cells. Concomitantly, an increase in the serum levels of interleukin-12 was observed, as well as an increased in the number of activated myeloid dendritic cells producing this cytokine. Moreover, a strong decrease in the survival was observed when Natural Killer cells were depleted using anti-asialo GM1 antibodies or following neutralization of interferon-γ. Thus, the host protection elicited by anti-CD20 treatment involves a network of cells from both innate and adaptive immunity that both are needed for long-term protection. In conclusion, our work demonstrates that anti-CD20 therapy promotes a strong anti-tumor adaptive immunity, opposes Treg expansion, and that a pro-tumor immune contexture shaped by the presence of growing tumor cells can be reversed by antibody treatment.

Citation Format: Claire Deligne, Sophie Siberil, Jean-Luc Teillaud. Anti-CD20 therapy prevents protumor regulatory T-cell expansion and triggers a memory Th1 response in tumor-bearing mice. [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 3636. doi:10.1158/1538-7445.AM2014-3636
Abstract 3656: MDSC-generated nitric oxide leads to an impairment of NK cell functions in metastatic melanoma patients
Authors: Trikha Prashant , Mundy-Bosse Bethany , Landi Ian , McMichael Elizabeth , Harper Amanda , Duggan Megan , Stasik Nancy , Kendra Kari , Olencki Thomas , Carson William
Published: May 2015

Myeloid-derived suppressor cells (MDSCs) are able to inhibit the function of immune cells within the tumor microenvironment. The interaction of MDSCs with T cells has been studied extensively. However, the interaction of MDSCs with innate immune cells such as NK cells is not well understood. This study investigated whether the presence of MDSCs could impair the function of NK cells.

MDSC (CD33+HLA-DRneg/lowCD11b+) and autologous NK cells (CD56+CD3neg) were isolated from the peripheral blood of metastatic melanoma patients and co-cultured overnight at a 1:1 ratio (2 x 105 cells each). Culture with MDSC led to a 70% decrease in NK cell mediated antibody dependent cellular cytotoxicity (ADCC) against HER2+ SkBr3 cells coated with an anti-HER2 antibody and a two-fold decrease in the production of interferon-gamma in response to these targets. Stimulation of the NK cell Fc receptor by immobilized immunoglobulin G (IgG) led to phosphorylation of Erk (p-Erk) and this event was significantly inhibited in the presence of MDSC in a dose and contact-dependent manner (p<0.05). Co-culture of NK cells with MDSC also led to reduced expression of the NKp30 activating receptor (1.7-fold decrease) and a decrease in cytotoxicity against the K562 cell line. MDSC were able to generate significant levels of nitric oxide (NO) and inhibition of MDSC NO synthesis in co-cultures led to a significant improvement in NK cell cytotoxicity and cytokine production (p<0.05). Depletion of MDSC from BALB/c mice bearing tumors of the CT26 adenocarcinoma cell line led to a three-fold increase in NK cell mediated ADCC. These results demonstrate that MDSC-derived NO can inhibit NK cell function, and suggest that strategies aimed at eliminating or deactivating MDSC may lead to enhanced efficacy of immune based therapies.

Citation Format: Prashant Trikha, Bethany Mundy-Bosse, Ian Landi, Elizabeth McMichael, Amanda Harper, Megan Duggan, Nancy Stasik, Kari Kendra, Thomas Olencki, William Carson. MDSC-generated nitric oxide leads to an impairment of NK cell functions in metastatic melanoma patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3656. doi:10.1158/1538-7445.AM2014-3656
Abstract 3660: Chronic alcohol consumption inhibits iNKT cell activation-induced antitumor response in B16BL6 melanoma-bearing mice
Authors: Zhang Hui , Zhang Faya , Zhu ZhaoHui , Meadows Gary G.
Published: May 2015

Alcohol consumption increases the incidence of many types of cancer including melanoma. It is not well understand how chronic alcohol consumption affects tumor progression and survival. We previously found that chronic alcohol consumption activates CD8+ T cells in the non-tumor injected mice and at the early stage after tumor inoculation; however, it accelerates CD8+T cell dysfunction during further tumor growth. The underlying mechanism regarding modulation of CD8+ T cells by alcohol and how the functional changes in the immune response affect tumor growth and survival remain to be elucidated. Herein, we investigated how alcohol modulates iNKT cells in melanoma-bearing mice and how different immune treatment regimens affect tumor growth and survival. We found that alcohol increases iNKT cells in non-tumor injected mice, and that activation of these iNKT cells induces a Th1 response. With tumor progression, alcohol reverses iNKT cell cytokine profile to aTh2-dominant one. Chronic alcohol consumption inhibits melanoma growth, but this is does associated with increased survival. Immunization with a melanoma cell lysate significantly inhibits tumor growth and increases survival compared to the non-immunized alcohol-consuming, tumor-bearing mice. Immunization in the water-drinking control mice did not significantly increase survival compared to respective non-immunized mice. Immunization coupled with iNKT cell activation with alpha GalCer significantly increases the survival of water-drinking melanoma-bearing mice. However, the survival of mice consuming alcohol was significantly decreased compared to their water-drinking counterparts. Collectively, chronic alcohol consumption interacting with melanoma alters the iNKT cell cytokine profile from Th1-dominant to Th2- dominant, which in turn contributes to inhibition of antitumor immunity and decreases survival of melanoma-bearing mice. Supported by NIH grants R01AA07293 and K05AA017149 and funds provided for medical and biological research by the State of Washington Initiative Measure No. 171.

Citation Format: Hui Zhang, Faya Zhang, ZhaoHui Zhu, Gary G. Meadows. Chronic alcohol consumption inhibits iNKT cell activation-induced antitumor response in B16BL6 melanoma-bearing mice. [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 3660. doi:10.1158/1538-7445.AM2014-3660
Abstract 3661: Crucial roles of cytokine-signaling for alteration in functions of myeloid-derived suppressor cells
Authors: Sumida Kentaro , Ono Yosuke , Ohtake Junya , Masuko Kazutaka , Terada Satoshi , Kaneumi Shun , Kishikawa Takuto , Kita Toshiyuki , Kitamura Hidemitsu
Published: May 2015

Various types of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) were generated from immature myeloid cells (ImCs) according to the state of tumor microenvironments. In human cancer patients, MDSCs are generally defined as monocytic (CD11b+, CD14+, and HLA-DR-/low) or granulocytic (CD11b+ and CD15+) myeloid cells with antitumor and immunosuppressive activities. In order to develop the effective cancer immunotherapy, these immune suppressor populations should be restrictlly controlled in tumor bearing hosts.

In the present research, we evaluated the immunological functions of monocytic or granulocytic myeloid cells, which were collected from tumor tissues of colorectal cancer patients. We found here that myeloid populations in tumor tissues significantly induced immuno-modulating factors such as IL-6, IL-10, VEGF, iNOS, ARG1, IDO1, and IDO2. In addition, we confirmed that tumor-infiltrating myeloid-populations exhibited a significant immunosuppressive activity than those prepared from peripheral blood mononuclear cells (PBMCs). To investigate how tumor-infiltrating MDSCs acquired the immunosuppressive activity, we focused on the effect of tumor-derived factors (TDFs) on immunosuppressive activity. PBMCs from healthy volunteers were cultured in the presence of IL-6, TGF-β, and granulocyte macrophage colony-stimulating factor (GM-CSF) for 6 days, and then differentiated into Mo-MDSCs (CD11b+CD14+CD33+)-like phenotype cells. The generated Mo-MDSCs were further isolated by using cell sorter system and cultured with donor matched T cells and stimulated with anti-CD3/CD28 beads for 3 days. As a result, we confirmed that T cell proliferation was significantly inhibited by the addition of Mo-MDSCs generated with IL-6, TGF-β and GM-CSF. On the other hand, these populations showed immunostimulating ability in the presence of IFN-g in vitro, suggesting the Mo-MDSCs might have multi-differencing potentials.

These results indicated that tumor-infiltrating MDSCs acquired stronger immunosuppressive activity by tumor-derived IL-6 and TGF-β, however the function might be altered in response to cytokine conditions. Thus, we concluded that IL-6 and TGF-β induced functional maturation of MDSCs in tumor microenvironments, suggesting promising targets to improve the immunological responses by immunotherapy for cancer patients.

Citation Format: Kentaro Sumida, Yosuke Ono, Junya Ohtake, Kazutaka Masuko, Satoshi Terada, Shun Kaneumi, Takuto Kishikawa, Toshiyuki Kita, Hidemitsu Kitamura. Crucial roles of cytokine-signaling for alteration in functions of myeloid-derived suppressor 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 3661. doi:10.1158/1538-7445.AM2014-3661
Abstract 367: Tumorigenesis: an investigation by network evolution and perturbations of somatic mutations in cancer interactome
Authors: Cheng Feixiong , Jia Peilin , Wang Quan , Lin Chen-Ching , Zhao Zhongming
Published: May 2015

Cells govern biological actions through highly complex biological networks. Perturbations to the complex molecular network due to driver mutations may transit cells to new phenotypic states, e.g., tumorigenesis. Identifying how genetic lesions such as somatic mutations perturb these networks is a fundamental challenge in cancer biology. The recent TCGA studies revealed that a typical tumor contains two to as many as eight of driver mutations while the numerous remaining somatic mutations are passenger mutations. So far, it remains largely unknown what evolutionary forces and how the genetic lesions disrupt cancer interactome that leads to tumorigenesis. In this study, we systematically investigated the relationship among the network topology, evolutionary rates, and evolutionary origins of somatic and germline mutation driven disease genes in the large context of the protein-protein interaction (PPI) networks by utilizing recently released extensive somatic mutations and gene annotation data. We aimed to address two fundamental questions. (1) Whether cancer genes display a distinct network topology from Mendelian disease genes, and why? (2) From a network biology perspective, how the transition occurs from a normal cell to a tumor cell as initiated by a few driver genetic mutations? We collected the largest ever cancer gene list and five comprehensive networks. We found evolutionary origin is the main determinant of the unique network centrality of cancer proteins. We further investigated the perturbations of network topology by somatic mutations that were identified from 3268 tumors across 12 cancer types in TCGA. We revealed that the network-attacking perturbation of somatic mutations on central hubs of cancer interactome is a main feature of tumor emergence and evolution. This finding elucidates the high efficiency of the transition during tumorigenesis as initiated by a few driver mutations. This work improves our understanding of dynamic network-attacking perturbation by somatic mutations during tumorigenesis, and, in turn, of the implications for both basic cancer biology and the development of personalized antitumor therapy.

Citation Format: Feixiong Cheng, Peilin Jia, Quan Wang, Chen-Ching Lin, Zhongming Zhao. Tumorigenesis: an investigation by network evolution and perturbations of somatic mutations in cancer interactome. [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 367. doi:10.1158/1538-7445.AM2014-367
Abstract 3676: Influence of early toxicology assessment on the selection of azd9291
Authors: Anderton Mark J. , Ward Richard A. , Daunt Paula , Galer Anne , Cross Darren A. E. , Marks Louise , Finlay M. Raymond V.
Published: May 2015

Discovery toxicology is the concept of evaluating the safety of molecules early in the drug development process when there is chemical scope to refine candidate drug quality. The aim is to identify and mitigate safety liabilities to maximize the chance of clinical success. This poster describes the impact of discovery toxicology on the selection of AZD9291, an irreversible inhibitor of both EGFR-TKI sensitising and resistance mutations. Although, maintaining a margin of selectivity to wild type EGFR was a key medicinal chemistry goal, early in vitro selectivity evaluation of the lead series compounds revealed significant off-target inhibition against the insulin and insulin-like growth factor receptors (IR/IGFR). Further exploration of this liability in a rat in vivo study revealed that our early lead compound (Cmpd 1) was associated with severe hyperglycemia, rapid body weight loss and evidence of reduced gastric emptying. The reduction in bodyweight and the inhibition of gastric emptying was dose limiting and we hypothesized this was caused by the hyperglycaemia. A broader selection of compounds based on the same scaffold was tested both in vitro and in vivo in the hope of identifying one or more compounds without this liability. Compounds were tested in a modified single dose rat study, exploring insulin and glucose homeostasis and effects on gastric emptying. Compounds within the shortlist caused increases in insulin of up to 80-fold, 3-fold increases in glucose and up to a 60% reduction in gastric emptying. There was a clear link between in vitro IR/IGF inhibition, hyperinsulinemia and inhibition of gastric emptying. One of the shortlisted candidates (now selected as AZD9291), had up to a 30 fold improvement in in vitro IGFR cellular potency compared to previous candidates and had no effect on insulin/glucose homeostasis in vivo. AZD9291 has demonstrated tumor shrinkage in patients at doses that have been very well tolerated and to date, no effect on glucose homeostasis has been reported.

Cmpd 1Cmpd 2Cmpd 3Cmpd 4Cmpd 5 (AZD9291)EGFRm+sensitising mutationcell IC50 (pEGF μM)0.390.0160.0210.0020.017EGFRm+/T790Mdouble mutationcell IC50 (pEGF μM)0.0910.0020.0040.00070.015EGFR wildtypecell IC50 (pEGF μM)23.00.360.940.150.48IGFRcell IC50 (pIGF μM)0.0990.160.490.103.3SM/IGFR selectivity0.25102348194

Citation Format: Mark J. Anderton, Richard A. Ward, Paula Daunt, Anne Galer, Darren A. E. Cross, Louise Marks, M. Raymond V. Finlay. Influence of early toxicology assessment on the selection of azd9291. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3676. doi:10.1158/1538-7445.AM2014-3676