bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3282: Induction and characterization of cyclopamine resistance in SK-N-DZ neuroblastoma and DAOY medulloblastoma cell lines
Authors: de la Rosa Javier , Paredes Amaia , Asensio-Salazar Javier , Fan Xing , Meléndez Bárbara , Rey Juan A. , Castresana Javier S.
Published: August 2015

Neuroblastoma and medulloblastoma are malignant solid tumors of childhood and early adolescence, that often reappear after clinical treatment due of an acquired chemo-resistance. These tumors present an increased expression of the Sonic Hedgehog signaling pathway. In the last years, inhibitors of this pathway, such as cyclopamine are being studied as a possible alternative to current treatment. In this research, we aim at analyzing the appearance and characteristics of resistance derived from the treatment with mutation-selective concentrations of cyclopamine. The aim of this study is the design of different ways to bypass the resistance generated by the tumor once its biology is completely understood.

Cells were exposed to several treatment cycles. A 24 h cyclopamine exposure was performed when cell culture reached 60-70% confluence. Basal half-maximal inhibitory concentration (IC50) was 10 μM. Hence, SK-N-DZ cells received 5 treatment cycles of cyclopamine at 12 μM dissolved in DMSO; whereas Daoy cells received 5 treatment cycles of cyclopamine at 10 μM. The control group only received the diluent.

Anchorage-independent growth assays (in soft agar), clonogenicity assays, cell migration scratch (wound-healing) assays, and relative mRNA expression were studied during cycles 1-5.

During the first 5 treatment cycles, mRNA expression of SMO remained constant. CD15 expression decreased. MYCN, Gli and expression showed an increasing trend. Decrease in the pro-apoptotic status was also described. Soft agar showed an increase tendency in relative colony formation capacity. No statistical differences were found in the clonogenicity assays for the SK-ND-Z cells.

The results highlighted the evidence that cells became resistant because they were able to grow in soft agar without attachment, to migrate and to overexpress genes related to cancer stem cells. Results shown are congruent with a resistance generation possibly owing to multidrug efflux pumps. However, more clear results would be seen with 48 h treatment. Further analysis based on this work could include 48 h treatment, water-dissolvable cyclopamine performance study, a more complete gene analysis, or studies with other neurological tumors.

Note: This abstract was not presented at the meeting.

Citation Format: Javier de la Rosa, Amaia Paredes, Javier Asensio-Salazar, Xing Fan, Bárbara Meléndez, Juan A. Rey, Javier S. Castresana. Induction and characterization of cyclopamine resistance in SK-N-DZ neuroblastoma and DAOY medulloblastoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3282. doi:10.1158/1538-7445.AM2015-3282
Abstract 3287: Targeting GSK-3: a new approach for the treatment of neuroblastoma
Authors: Dubrovskyi Oleksii , Ugolkov Andrey , Gaisina Irina , Bondarenko Gennadiy , Strizzi Luigi , Margaryan Naira , O'Halloran Thomas , Kozikowski Alan , Hendrix Mary , Mazar Andrew
Published: August 2015

Neuroblastoma is a devastating pediatric cancer and most patients older than 18 months present with multi-organ metastatic disease. High grade or recurrent disease is refractory to treatment with chemotherapy and almost uniformly fatal. Because GSK-3beta has been shown to be a positive regulator of NF-kappaB-mediated survival and chemoresistance in cancer cells, we hypothesize that the inhibition of GSK-3 may have potential therapeutic effects in neuroblastoma. To test this premise we used small molecule inhibitors, Western blotting, apoptotic and MTS assays to study the effect(s) of GSK-3 inactivation in neuroblastoma cell lines SK-N-DZ and SK-N-BE(2). Using chemically distinct GSK-3 inhibitors (AR-A014418, TDZD8 and 9-ING-41), we found that pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of neuroblastoma cells. We observed that inhibition of GSK-3 results in decreased expression of the anti-apoptotic molecules Bcl-XL and XIAP (NF-kappaB target genes) and a subsequent increase in neuroblastoma cell apoptosis. Moreover, we show that our novel GSK-3 inhibitor 9-ING-41 enhances the antitumor effects of irinotecan in neuroblastoma cells. Our results demonstrate that GSK-3 positively regulates neuroblastoma cell survival and proliferation and suggest that the inhibition of GSK-3 is a promising new approach for the treatment of neuroblastoma.

Citation Format: Oleksii Dubrovskyi, Andrey Ugolkov, Irina Gaisina, Gennadiy Bondarenko, Luigi Strizzi, Naira Margaryan, Thomas O'Halloran, Alan Kozikowski, Mary Hendrix, Andrew Mazar. Targeting GSK-3: a new approach for the treatment of neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3287. doi:10.1158/1538-7445.AM2015-3287
Abstract 3288: The KCNA5 promoter is hypermethylated in Ewing sarcoma and silencing contributes to cell proliferation
Authors: Ryland Katherine , Hawkins Allegra , Weisenberger Daniel J. , Borinstein Scott , Laird Peter W. , Martens Jeffrey R. , Lawlor Elizabeth R.
Published: August 2015

Potassium channels regulate a variety of biological processes and dysfunction of these channels contributes to cancer initiation and progression. We recently showed that the KCNA5-encoded channel, Kv1.5, is a target of polycomb-group (PcG)-dependent repression in aggressive pediatric solid tumors and that this epigenetic silencing of the Kv1.5 channel contributes to cell survival under hypoxic stress. In human cancer, the promoters of PcG target genes are often subject to aberrant DNA hypermethylation. Therefore, in the current study we investigated whether the KCNA5 locus is hypermethylated in Ewing sarcoma. The methylation status of KCNA5 was determined in tumor samples, non-transformed cells and a panel of cancer cell lines using a custom Illumina GoldenGate® methylation array and by MethyLight. Promoter methylation and expression of KCNA5 were measured by MethyLight and qRT-PCR, respectively, before and after exposure to decitabine. Proliferation was assessed in untreated and decitabine treated cells in the presence or absence of the Kv1.5 channel blocker diphenyl phosphine oxide-1 (DPO-1). Methylation array studies of normal tissues and Ewing sarcoma tumors and cell lines showed that the KCNA5 promoter is hypermethylated in Ewing sarcoma compared to normal tissues. This hypermethylation was confirmed by MethyLight analysis in an independent set of primary tumors and cell lines. These studies also showed that methylation was relatively higher in cell lines than primary tumors. Exposure of Ewing sarcoma cells to decitabine led to demethylation of the KCNA5 promoter and directly correlated with increased expression of the KCNA5 transcript. Decitabine treatment did not affect the viability of Ewing sarcoma cell lines; however it led to a decreased proliferation. Significantly, pharmacologic inhibition of the Kv1.5 channel by DPO-1 partially reversed the decitabine-induced reduction in cell proliferation in three different cell lines (TC-71, A673 and A4573). Together these studies show that the KCNA5 promoter is hypermethylated in Ewing sarcoma and that inactivation of the Kv1.5 channel promotes cell proliferation. This work identifies DNA methylation as another epigenetic mechanism that regulates KCNA5 expression in cancer and further implicates epigenetic repression of the Kv1.5 potassium ion channel in tumor pathogenesis.

Citation Format: Katherine Ryland, Allegra Hawkins, Daniel J. Weisenberger, Scott Borinstein, Peter W. Laird, Jeffrey R. Martens, Elizabeth R. Lawlor. The KCNA5 promoter is hypermethylated in Ewing sarcoma and silencing contributes to cell proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3288. doi:10.1158/1538-7445.AM2015-3288
Abstract 332: Extracellular matrix stiffness regulates metabolic state in metastatic, but not quiescent, breast carcinoma cells
Authors: Morris Brett A. , Burkel Brian , Ponik Suzanne , Eliceiri Kevin , Aguirre-Ghiso Julio , Condeelis John , Castracane James , Keely Patricia J.
Published: August 2015

Increased breast density is associated with a 4-6 fold increased risk of developing breast cancer, and is associated with an increase in deposition of extracellular matrix (ECM) proteins, most abundantly collagen I. Collagen, like other ECM proteins, plays a structural role fundamental to tissue organization. High levels of collagen deposition correspond with a stiffer ECM, which is emerging as an important regulator of cell proliferation and tumor progression. Our previously reported microarray implicated changes in mammary epithelial cell metabolism in response to increased matrix stiffness, consistent with the expanding role of the ECM in tumor cell signaling. Here we report that increased matrix stiffness regulates the expression of pyruvate dehydrogenase kinase 1, a key regulator between lactic acid production and pyruvate entry into the mitochondria, in highly metastatic 4T1 breast carcinoma cells. Interestingly, we do not observe this same metabolic regulation in quiescent (dormant) 4T07 tumor cells of the same lineage. These alterations in protein expression correlated to changes in cellular NADH metabolism observed by several independent approaches, including metabolic flux analysis and quantitative imaging fluorescence lifetime microscopy. Thus, we find that alterations in collagen stiffness cause metabolic shifts between oxidative phosphorylation and aerobic glycolysis in highly metastatic cells, but not in quiescent cells. These findings identify stiffness of the ECM as an important regulator of metabolic state, and further identify quiescence as a dominant trait that is not overcome by ECM stiffness.

Citation Format: Brett A. Morris, Brian Burkel, Suzanne Ponik, Kevin Eliceiri, Julio Aguirre-Ghiso, John Condeelis, James Castracane, Patricia J. Keely. Extracellular matrix stiffness regulates metabolic state in metastatic, but not quiescent, breast carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 332. doi:10.1158/1538-7445.AM2015-332
Abstract 3324: A novel role for PAK4 in the regulation of EMT and stemness in glioblastoma
Authors: Kesanakurti Divya , Xu Jihong , Jasti Rao S. , Puduvalli Vinay K.
Published: August 2015

Glioblastomas (GB) are the most aggressive of primary brain tumors with high rates of tumor recurrence and poor prognosis. Epithelial-mesenchymal transition (EMT) is the dynamic transdifferentiation process which leads to acquisition of invasiveness, stem-like characteristics and drug resistance in tumor cells. A small fraction of radio- and chemo-resistant cancer stem cells (CSC) with a capacity of self-renewal is considered to be crucial for tumor recurrence. Our previous studies indicated high levels of expression of p21-activated kinase 4 (PAK4) in GBs and confirmed its role in the regulation of proliferation, migration and invasion. Here, we aimed to determine whether PAK4-regulated invasiveness was associated with mesenchymal transition of glioma cells and to delineate the essential signaling events that governed EMT and maintenance of stemness in GBs. PAK4 overexpression in glioma cells resulted in loss of E-cadherin and Zo-1 and a significant increase in N-cadherin and vimentin levels. Conversely, shRNA-mediated PAK4 downregulation led to loss of mesenchymal phenotype and markers in glioma cell lines. Further, irradiation (IR, 8 Gy) treatments significantly elevated PAK4 expression and nuclear localization in correlation with increased EMT in glioma cells. FACS analysis and western blotting indicated an enrichment of CD133+ CSCs with high expression of CD133 and Nestin in IR-treated cells. In order to study the significance IR-induced EMT and stemness with PAK4 nuclear translocation, we performed a transcription factor (TF) interaction array with immunoprecipitated PAK4 as bait, which revealed nuclear-PAK4 association with PPARγ and other TFs. Moreover, immunoprecipitation, ChIP and re-ChIP experiments showed IR-induced PAK4 binding with PPARγ nuclear receptor complex and recruitment on the PPREs of NADPH oxidase-1 promoter and elevated Nox1 expression in IR-treated cells. PAK4-knockdown suppressed the IR-induced EMT and stemness in the glioma cells lines. In line with these in vitro results, mouse orthotopic intracranial xenograft experiments showed decreased tumor growth and N-cadherin expression in tumors from PAK4-shRNA expressing cells when compared with controls. These results strongly support a role for PAK4 in the regulation of IR-induced EMT and stemness via PPARγ/Nox1 signaling and provide a rationale for PAK4 as a new therapeutic target against glioblastoma and suggest a potential for overcoming radioresistance in these tumors.

Citation Format: Divya Kesanakurti, Jihong Xu, Rao S. Jasti, Vinay K. Puduvalli. A novel role for PAK4 in the regulation of EMT and stemness in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3324. doi:10.1158/1538-7445.AM2015-3324
Abstract 3328: Mitigating long-term cardiac adverse effects of radiation exposure: Emerging opportunities in protein oxidation and autophagy modulation
Authors: Rosen Elliot T. , Kryndushkin Dmitry , Gonzalez Yanira , Aryal Baikuntha , Chehab Leena , Dickey Jennifer , Rao Ashutosh
Published: August 2015

Radiation-induced heart disease (RIHD) presents a significant challenge in the oncology setting. Patients who receive acute doses of irradiation to the thoracic cavity for Hodgkin's Lymphoma and breast cancer are subject to an elevated risk of RIHD. The ability to mitigate the long-term cardiac effects of radiation exposure in these populations by modulating protein oxidation and the process of autophagy in the heart has the potential to lead to improved health outcomes. We utilized the spontaneously hypertensive rat (SHR), which has proven a good model for drug-induced cardiotoxicity, to evaluate radiation-induced heart disease. SHRs were exposed to low, medium, or high doses of full-body irradiation. Necropsy was performed to collect serum and tissue samples at various time points up to one year. Echocardiography and electrocardiography were performed to evaluate functional changes in cardiac performance. Four weeks following ionizing radiation, both males and females showed decreased heart mass and decreased whole body mass in the groups exposed to medium and high doses of radiation. A marked difference in both heart and whole body mass was observed in the high dose group after one year. Anemic conditions were noted by low red blood cell counts observed in both males and females at all three time points in the animals receiving the highest dose of irradiation. Cardiac troponin T analysis revealed cardiotoxicity in both the medium and high dose groups at early time points. Echocardiography revealed that left ventricular volume at diastole was increased in males following high dose irradiation, and that left ventricular mass decreased following high irradiation exposures. Finally, it should also be noted that after one year, multiple animals receiving radiation developed spontaneous tumors. We are currently exploring the oxidative stress-mediated molecular mechanisms behind the radiation-induced cardiac damage and the subsequent, protective autophagic response.

Citation Format: Elliot T. Rosen, Dmitry Kryndushkin, Yanira Gonzalez, Baikuntha Aryal, Leena Chehab, Jennifer Dickey, Ashutosh Rao. Mitigating long-term cardiac adverse effects of radiation exposure: Emerging opportunities in protein oxidation and autophagy modulation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3328. doi:10.1158/1538-7445.AM2015-3328
Abstract 3349: Optimizing radioimmunotherapy techniques for the alpha-emitter 225Actinium
Authors: Julian Ricklie A. , Kang Chi Soo , Sun Xiang , Song Hyun A. , Revskaya Ekaterina , Chong Hyun-Soon , Dadachova Ekaterina
Published: August 2015

Introduction: Radioimmunotherapy (RIT) is a potent therapeutic technique that may be applicable to the treatment of numerous human cancers. Currently available RIT therapies employ beta- and alpha-emitting radionuclides. Alpha-emitters, such as 225Actinium (225Ac), have promising advantages over beta-emitters RIT such high linear energy transfer and independence of the tumor oxygenation status. RIT using 225Ac radionuclide directed by the monoclonal antibodies trastuzumab or panitumumab may provide effective, targeted antineoplastic effects.

Methods: Antibodies trastuzumab to HER2/neu and panitumumab to EGFR were conjugated to the bifunctional ligands NETA, DOTA, DECA, DEPA and DETA. The conjugates were labeled with 225Ac at room temperature and 37°C. At selected time points, the mixture was analyzed by instant thin layer chromatography (ITLC). Radiolabeling reaction kinetics and yields were calculated for 225Ac and for its daughter isotopes 221Francium (221Fr) and 213Bismuth (213Bi) using the energy discrimination function of the multichannel analyzer in the gamma counter. Summary of the new data: Radiolabeling with Actinium was effective for several ligand-antibody combinations and completed within 30 min. Radiolabeling yields for 225Ac and it daughters 221Fr and 213Bi were most robust for NETA and DECA conjugates to trastuzumab and panitumumab, labeled at 37°C.

Conclusion: Several novel ligands were evaluated for radiolabeling of monocloncal antibodies trastuzumab or panitumumab with 225Ac radionuclide and demonstrated fast kinetics of radiolabeling. Potential for targeted therapeutic applications will be explored in future experiments.

Citation Format: Ricklie A. Julian, Chi Soo Kang, Xiang Sun, Hyun A. Song, Ekaterina Revskaya, Hyun-Soon Chong, Ekaterina Dadachova. Optimizing radioimmunotherapy techniques for the alpha-emitter 225Actinium. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3349. doi:10.1158/1538-7445.AM2015-3349
Abstract 3352: Identification of factors that recruit tumor-associated neutrophils to head and neck squamous cell cancer
Authors: Niaz Hanan A.
Published: August 2015

Recent immunohistological studies have shown that abundant tumour-associated neutrophils (TAN) are present within malignant tumours. TAN can have a significant impact on the tumour microenvironment via release of potent growth factots, chemokines and proteinases that influence tumour progression. The presence of TAN has been associated with poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC). The aims of this study were to identify which factors are responsible for neutrophil recruitment into HNSCC.

The number of TAN within human HNSCC tissue was evaluated by immunohistochemical staining for the neutrophil marker, myeloperoxidase (MPO). The factors secreted by FaDu HNSCC multi-cellular tumour spheroids (MCTS) were analysed by cytokine array and ELISA. Neutrophils isolated from the peripheral blood of healthy volunteers were used to measure neutrophil migration to factors identified from the array. Then the recruitment of neutrophils to MCTS was assessed overtime by flow cytometry in the absence and presence of small molecule inhibitors. FaDu xenograft mouse models were used to confirm the effect of these inhibitors on neutrophil recruitment in-vivo.

MPO staining confirmed the presence of marked numbers of TAN in HNSCC compared to normal oral epithelium. HNSCC MCTS resemble in-vivo tumours, displaying areas of hypoxia, necrosis and cell proliferation. Neutrophils migrated to recombinant CXCL8, CXCL1 and MIF and these chemoattractants were found in the conditioned medium of FaDu MCTS. The recruitment of neutrophils to FaDu MCTS was significantly inhibited when neutrophils were pre-treated with antagonists for CXCR2 and CXCR4, the receptors for CXCL8, CXCL1 and MIF respectively. Moreover, use of the MIF inhibitor, ISO-1caused a dramatic reduction in the number of neutrophils recruited into FaDu MCTS. In-vivo, ISO-1 significantly reduced the number of TAN in xenograft FaDu tumours. Collectively, these data suggest that CXCL8, CXCL1 and MIF in particular are important in the recruitment of TAN into HNSCC.

Citation Format: Hanan A. Niaz. Identification of factors that recruit tumor-associated neutrophils to head and neck squamous cell cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3352. doi:10.1158/1538-7445.AM2015-3352
Abstract 3358: SOCS3 deficiency in myeloid cells promotes tumor development: Involvement of STAT3 activation and myeloid-derived suppressor cells
Authors: Yu Hao , Qin Hongwei , Benveniste Etty (Tika)
Published: August 2015

Suppressor Of Cytokine Signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway, and generally function as tumor suppressors. The absence of SOCS3 in particular leads to heightened activation of the STAT3 transcription factor, which has a striking ability to promote tumor survival while suppressing anti-tumor immunity. We report for the first time that genetic deletion of SOCS3 specifically in myeloid cells significantly enhances tumor growth, which correlates with elevated levels of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment, and diminished CD8+ T-cell infiltration in tumors. Furthermore, SOCS3-deficient bone-marrow-derived cells exhibit heightened STAT3 activation and preferentially differentiate into the Gr-1+CD11b+Ly6G+ MDSC phenotype. Importantly, we identify granulocyte-colony stimulating factor (G-CSF) as a critical factor secreted by the tumor microenvironment that promotes development of MDSC via a STAT3-dependent pathway. Abrogation of tumor-derived G-CSF reduces the proliferation and accumulation of Gr-1+CD11b+ MDSC and inhibits tumor growth. These findings highlight the critical function of SOCS3 as a negative regulator of MDSC development and function, via inhibition of STAT3 activation.

Note: This abstract was not presented at the meeting.

Citation Format: Hao Yu, Hongwei Qin, Etty (Tika) Benveniste. SOCS3 deficiency in myeloid cells promotes tumor development: Involvement of STAT3 activation and myeloid-derived suppressor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3358. doi:10.1158/1538-7445.AM2015-3358
Abstract 3362: Systematic evaluation of immune checkpoint inhibitors
Authors: Barnes Sheri , Miliani de Marval Paula , Hauser Jacob , Brainard Tracy , Small Daniel , Synnott Aidan J. , Mullin Robert J.
Published: August 2015

With the increasing success and subsequent interest in tumor immunology, we recognized a growing need for well-characterized preclinical models. While the literature contains many different experimental models, the available data includes a variety of reagents and the side by side comparative evaluation of therapeutics in multiple models is relatively rare. Lechner et al., (J. Immunother. 36:477-489, 2013) addressed this issue and reported on a comparative analysis of a set of six syngeneic models. Our effort looked to expand this paradigm towards a comparative evaluation of the responsiveness of a collection of syngeneic models to antibody based checkpoint inhibitor therapeutics. Specifically, we examined the response of the Colon26, MC38, B16F10, Lewis Lung, Madison109, EMT-6, and 4T1 models to anti-CTLA-4, anti-PD-1 and anti-PDL-1 monotherapies as well as capturing the response to combination therapies of anti-CTLA-4 with anti-PD-1 or anti-PDL-1.. Our results clearly show a differential response across this set of models both in regard to CTLA-4 targeted therapy as well as PD-1/PDL-1 therapy. In an attempt to expand the number of checkpoint inhibitor responsive models, we are performing a second phase of work evaluating the impact of immuno-modulatory radiation therapy upon these baseline responses.

In initiating these studies and evaluating the literature, it became clear that there were multiple choices in regard to anti-CTLA-4 preclinical reagents. We have also performed a series of studies evaluating the efficacy of multiple anti-CTLA-4 clones as an extension of previously published work which described the variable ability of anti-CTLA-4 clones to deplete tumor Treg populations (Simpson et al. J. Exp. Med. 210(9) 1695-1710, 2013). Our anti-tumor data correlates well with these cell based observations. This differential allows one to match efficacy with model and expands the reagents available for evaluating combination therapies.

Citation Format: Sheri Barnes, Paula Miliani de Marval, Jacob Hauser, Tracy Brainard, Daniel Small, Aidan J. Synnott, Robert J. Mullin. Systematic evaluation of immune checkpoint inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3362. doi:10.1158/1538-7445.AM2015-3362
Abstract 3366: The mechanical microenvironment and β1/FAK signaling control fibroblast accumulation in lung cancer
Authors: Alcaraz Jordi , Gabasa Marta , Busnadiego Óscar , Rodríguez-Pascual Fernando , Gascón Pere , Reguart Noemí
Published: August 2015

There is growing interest in targeting the abnormal accumulation of fibroblasts in solid tumors. The current paradigm assumes that the abundance of tumor associated fibroblasts (TAFs) is largely driven by soluble growth factors as in generic repair responses irrespective of the tumor type or subtype. We recently challenged this assumption in cultured TAFs from two major subtupes of non-small cell lung cancer (NSCLC): adenocarcinoma (ADC) and squamous cell carcinoma (SQC). We reported that the increased population of SQC-TAFs is largely driven by matrix stiffening rather than by soluble growth factors. In contrast, ADC-TAFs were poorly responsive to exogenous mechanical cues. We also reported that the differential mechanoresponses in SQC- and ADC-TAFs were associated with both larger mechanosensing through FAK and β1 integrin expression in the former TAFs. To get more insights into the latter mechanoregulatory circuit, we forced either β1 integrin activation or FAK expression in fibroblasts cultured in soft (normal-like) gels. Both conditions were sufficient to increase fibroblast population to values comparable to those found in stiff (tumor-like) gels. These data reveal that an abnormally high intrinsic mechanosensing through β1 integrins and FAK can bypass the inhibitory (protective) role of an extrinsic soft microenvironment. Moreover, these results suggest that inhibiting either β1 integrin or FAK signaling may be a suitable approach to target tumor-supporting TAFs in SQC.

Citation Format: Jordi Alcaraz, Marta Gabasa, Óscar Busnadiego, Fernando Rodríguez-Pascual, Pere Gascón, Noemí Reguart. The mechanical microenvironment and β1/FAK signaling control fibroblast accumulation in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3366. doi:10.1158/1538-7445.AM2015-3366
Abstract 3370: Incorporation of mesenchymal stem cells into areas of lung metastasis in an osteosarcoma model
Authors: Gutierrez Luciana M. , Amoros Mariana A. , Sevlever Gustavo , Podahjcer Osvaldo , Cremaschi Graciela , Kleinerman Eugenie S. , Bolontrade Marcela F.
Published: August 2015

Lung is the most common metastatic site in osteosarcoma patients with a majority presenting lung micrometastasis at diagnosis time. MSCs compose a heterogeneous cell population with the ability to migrate and home into remodeling tissues, including tumors. To evaluate the participation of MSCs in lung metastasis formation, we intravenously injected LM7 osteosarcoma cells into immunosuppressed mice. Pre-labelled MSCs (DiR+/CMDiI+, at a dose of 0.5-1×10e6 cells) were intravenously administered 10 weeks after tumor cells injection, when microscopic metastasis become apparent. MSCs biodistribution was evaluated both through real time (DiR+) and microscopic (CMDiI+) analysis. MSCs levels in the lungs of tumor-bearing mice as compared to mice with no tumor burden was 1.15×10E5±0.07 p2/sec/cm2/sr vs. 5.44×10E4±0.5p2/sec/cm2/sr (one week post-inoculation, infrared signal, p<0.01). This indicates a high homing ability of the stromal cells into the metastatic tumor microenvironment. Microscopic analysis corroborated infrared signal with MSCs presence and incorporation within the metastatic sites. In addition, MSCs displayed heterogeneous distribution within lungs with LM7 metastasis as opposed to the homogeneous MSCs distribution observed in lungs without metastasis. This suggests that MSCs are chemoattracted and retained in preferential areas within the lungs with LM7 osteosarcoma metastases. The setup of an experimental osteosarcoma metastatic model which incorporates administered MSCs as tumor stromal cells will be useful to analyze MSCs function in tumor metastasis formation.

Note: This abstract was not presented at the meeting.

Citation Format: Luciana M. Gutierrez, Mariana A. Amoros, Gustavo Sevlever, Osvaldo Podahjcer, Graciela Cremaschi, Eugenie S. Kleinerman, Marcela F. Bolontrade. Incorporation of mesenchymal stem cells into areas of lung metastasis in an osteosarcoma model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3370. doi:10.1158/1538-7445.AM2015-3370
Abstract 3373: Identification of inflammation-related genes that regulate tumor-associated stemness using high-throughput siRNA screening
Authors: Xie Junling , He Huiwen , Chen Chong , Bai Lipeng , Wang Wei , Liu Yan , Guo Jian , Wu Peng , Xiang Rong , Luo Yunping
Published: August 2015

Cancer associated stem cells (CSCs) are considered to be responsible for tumor recurrence, metastasis as well as chemoresistance. These properties of tumor associated stemness are maintained by specialized microenvironment, including inflammation which is a key component of the tumor microenvironment. To study the relationship between inflammation and stemness of CSCs, here, we established a robust high-throughput RNAi screen platform for a global survey of inflammation-related genes affecting CSCs identity via alteration of Oct4 expression. Consequently, we found 72 novel genes from 1027 inflammation-related genes which could significantly affect OCT4 expression. These genes are functionally distinct including cytokines, adhesion molecules, apoptotic molecules, kinase, metabolism-related enzymes and transcriptional factors. Further, we carry out a functional validation of the 72 hits by mean of ALDEFLUORTM assay and we demonstrate that silence genes expression of IL-1A, IL-1B, ICAM3, CCL16, TRAF6, PRTN3, PDE3A, BCAR1, NFKB1 and IKK-β by siRNA would dramatically downregulate of the ALDH+ subpopulation cells. Moreover, we studied the functional roles of these factors on different kinds of tumor cell lines and mult-organ tumor tissue microarray. We observed that knockdown TRAF6 (TNF-α receptor associated factor 6) would reduce the expression of stemness associated genes SOX2, OCT4, NANOG, LIN28b, IKK-β β-catenin, and abate sphere-forming ability of breast cancer MDA-MB-231 cells, lung cancer A549 cells, liver cancer HepG2 cells and colon cancer SW-480 cells. It was also found that the expression of TRAF6 has a conspicuous relationship with multiple tumor occurrence and malignancy on tissue microarray. Most importantly, we found that knockdown the expression of TRAF6 could markedly suppressed tumor growth and metastasis of MDA-MB-231 in NOD/SCID mice model. Together, we uncovers a wealth of novel inflammatory regulators of CSCs by a high-throughput siRNA screen and validated the cytokine TRAF6 could govern tumor associated stemness in different tumors in vitro and in vivo, which would be a potential therapeutical target.

Citation Format: Junling Xie, Huiwen He, Chong Chen, Lipeng Bai, Wei Wang, Yan Liu, Jian Guo, Peng Wu, Rong Xiang, Yunping Luo. Identification of inflammation-related genes that regulate tumor-associated stemness using high-throughput siRNA screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3373. doi:10.1158/1538-7445.AM2015-3373
Abstract 3378: Discoidin domain receptor 2 differentially controls HT-1080 cell proliferation in young-adult and old type I collagen 3D matrices
Authors: Saby Charles , Garnotel Roselyne , El Btaouri Hassan , Van Gulick Laurence , Jeannesson Pierre , Morjani Hamid
Published: August 2015

Type I collagen plays a key role in tumor proliferation, adhesion and migration. We have recently demonstrated that this fibrillar and life-long protein undergoes detrimental changes in biochemical and biomechanical properties during aging (1). In addition to a high Advanced Glycation End-products (AGE) and cross-links content, old collagen exhibits a significant loss in fibrillar properties.

In this study, we analyzed the effect of collagen aging on human HT-1080 fibrosarcoma cell proliferation in 3D matrix model. For this, type I collagen was extracted from tail tendons of young-adult (2 months) and old (2 years) rats. In 3D matrix, the rate of HT-1080 cell growth was significantly lower in young-adult collagen. This was accompanied by a down-regulation of ERK1/2 phosphorylation and an increase in the cyclin-dependent kinase inhibitor p21 expression. Blocking antibodies and siRNA strategy against β1 integrin and AGEs receptor (RAGE) did not increase cell growth in young-adult collagen.

Accumulating evidence suggest that Discoidin Domain Receptor 2 (DDR2) is a receptor tyrosine kinase with the unique ability to be activated by fibrillar collagen. To determine whether differential DDR2 responses are elicited by the two collagens, DDR2 was immunoprecipitated from cell lysates, and its phosphorylated form was detected by immunoblotting. DDR2 phosphorylation level was increased in young-adult collagen. This was accompanied by an increase in the tyrosine phosphatase SHP2 phosphorylation, the primary target of DDR2. Interestingly, the DDR2 inhibitor nilotinib was able to restore cell proliferation in young-adult collagen. In addition to an increase in ERK1/2 phosphorylation, nilotinib decreased SHP2 phosphorylation and p21 expression.

Taken together, these data show that age-associated changes of type I collagen organization, especially fibrillar properties, are critical for tumor cell proliferation control by DDR2.

References: (1) Wilson SL et al. FASEB J. 2014 28(11):14-25

Citation Format: Charles Saby, Roselyne Garnotel, Hassan El Btaouri, Laurence Van Gulick, Pierre Jeannesson, Hamid Morjani. Discoidin domain receptor 2 differentially controls HT-1080 cell proliferation in young-adult and old type I collagen 3D matrices. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3378. doi:10.1158/1538-7445.AM2015-3378
Abstract 3388: Analytical validation and clinical verification of phosphoprotein biomarker modulation using a novel preservation system-based flow cytometry assay in multiple myeloma clinical trials
Authors: Pahuja Anil , Sarikonda Shyam , Lee Benjamin , Graber Armin , Tangri Shabnam , Dakappagari Naveen
Published: August 2015

Propelled by the advent of novel oncogenic pathway-targeting drugs and increasing regulatory expectations to fulfil proof of mechanism endpoints in clinical trials, there is a critical need to reliably evaluate target inhibition by investigational drugs in biologically relevant compartments. However, the ability to monitor modulation of labile oncogenic pathway biomarkers in global hematomalignancy clinical trials is complicated by varying technical expertise at clinical sites, when specimen stability considerations do not permit analysis at a central specialty laboratory. To address this critical gap, we developed a novel preservation system, designated NovaPerm3 (NP3), that enables stabilization of phosphoprotein based biomarker specimens in a single step, requires less than 20 minutes, and can be implemented in routine clinical trial settings. Stabilized specimens can be frozen and analyzed even after 90 days in a specialized laboratory. Using the NP3 preservation system, we systematically developed and validated a flow cytometry assay that not only enables reliable identification of neoplastic plasma cells but also allows for quantitation of phosphoprotein biomarkers in the tumor cells of interest present in both the peripheral blood (PB) and bone marrow (BM). We will describe a) modulation of phosphorylation levels of the S6 ribosomal protein (pS6RP) by a novel anticancer agent in multiple myeloma patients b) qualitative concordance between NP3-flow cytometry and western blot results from multiple drug treated leukemia cell lines c) sensitive detection of neoplastic plasma cells up to 1% of the total white cells and d) specimen (pS6RP) stabilization over a 90 day period. In conclusion, our novel preservation method and clinical trial laboratory validated flow assay enables reliable quantification of signaling biomarkers in hematomalignancy trials with ease in a specialized central laboratory while reducing the procedural burden for collecting biomarker specimens in global clinical trials.

Citation Format: Anil Pahuja, Shyam Sarikonda, Benjamin Lee, Armin Graber, Shabnam Tangri, Naveen Dakappagari. Analytical validation and clinical verification of phosphoprotein biomarker modulation using a novel preservation system-based flow cytometry assay in multiple myeloma clinical trials. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3388. doi:10.1158/1538-7445.AM2015-3388
Abstract 339: Different receptor tyrosine kinases mediate EMT and erlotinib-resistance in NSCLC cell lines through bypass signalling
Authors: Raaby Jakobsen Kristine , Demuth Christina , Madsen Anne Tranbjerg , Westh Mortensen Birgit , Meldgaard Peter , Lade Nielsen Anders , Sandahl Soerensen Boe
Published: August 2015

Introduction: Erlotinib is a potent drug in the treatment of EGFR-driven lung cancer. Many patients initially benefit from erlotinib treatment, but nearly all acquires resistance and die of the disease. Several mechanisms related to erlotinib-resistance have been described including epithelial to mesenchymal transition (EMT). Signalling by TGFβ, Wnt or through receptor tyrosine kinases can initiate EMT, but not much is known about the trigger in relation to erlotinib-resistance. In this study, we investigate how EMT is initiated in two erlotinib-sensitive NSCLC cell lines developing erlotinib-resistance.

Methods: HCC827 (EGFR mutated) and Calu-3 (HER2 amplified) were treated with erlotinib in escalating concentrations (0.01 μM - 5 μM) over approximately 3 months, and erlotinib resistant (ER) sublines were established and investigated.

Results: Both HCC827ER and Calu-3ER gained EMT features including upregulation of SLUG, SNAIL and ZEB1. These changes were accompanied by increased IGF1R signalling in Calu-3ER and HCC827ER gained a MET amplification. To detect the relevance of MET and IGF1R signalling in accordance to EMT, we treated the cells with the tyrosine kinase inhibitors crizotinib (MET) and linsitinib (IGF1R). In both cases, we saw a decrease in EMT-marker transcription after the treatment. In addition, HCC827ER showed growth inhibition when treated with the MET-inhibitor crizotinib, but linsitinib did not inhibit the growth of Calu-3ER. Conclusion: Our study demonstrates how different receptor tyrosine kinase signalling pathways can induce EMT during the development of erlotinib-resistance. Furthermore, we show that EMT can accompany well-know resistance mechanisms such as MET-amplification and hereby contribute to the phenotype of the resistant cells.

Citation Format: Kristine Raaby Jakobsen, Christina Demuth, Anne Tranbjerg Madsen, Birgit Westh Mortensen, Peter Meldgaard, Anders Lade Nielsen, Boe Sandahl Soerensen. Different receptor tyrosine kinases mediate EMT and erlotinib-resistance in NSCLC cell lines through bypass signalling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 339. doi:10.1158/1538-7445.AM2015-339
Abstract 3404: NDRG2 suppresses growth and invasion of gallbladder carcinoma via regulation of matrix metalloproteinase 19/slug interaction
Authors: Kim Nayoung , Min Jeong-Ki , Kim Jin-Man , Lee Hyo Jin
Published: August 2015

Background: Gallbladder cancer is a relatively uncommon tumor worldwide but it has a feature of highly invasive and malignant tumor with poor prognosis. However, relevant molecules and/or genes associated with growth and metastasis of gallbladder carcinoma are poorly understood. Although NDRG2 is known as a tumor suppressor gene, its exact role in gallbladder carcinoma remains unknown.

Materials and method: For deciphering biological roles of NDRG2 in gallbladder carcinoma, first, we examined the expression of NDRG2 in tumor specimens from 86 gallbladder carcinoma patients by immunostaining and analyzed the correlation between NDRG2 expression and clinicopathologic parameters as well as patient survival. Second, we investigated the functional significance of NDRG2 in gallbladder carcinoma progression by stable knock-down or overexpression of NDRG2 in in vitro and in vivo studies.

Results: NDRG2 was differentially expressed in gallbladder carcinoma tissues. The negative expression of NDRG2 was significantly associated with a pathological T stage (P = 0.015), high histological grade (P = 0.020), and perineural invasion (P = 0.016). Multivariate analyses clearly showed that the loss of NDRG2 expression was an independent poor prognostic factor for cancer-specific survival. The stable expression of NDRG2 shRNA in gallbladder carcinoma cell lines significantly increased cell proliferation, anchorage-independent growth, migration and invasion. In addition, NDRG2 shRNA remarkably increased metastasis from the spleen to the liver in mice. Furthermore, loss of NDRG2 expression in cells caused an epithelial-mesenchymal transition through Slug induction by MMP-19 interaction with Slug promoter.

Conclusions: In the present study, we showed that loss of NDRG2 expression leads to poor prognosis and enhanced metastasis in gallbladder carcinoma. NDRG2 suppresses MMP-19 which causes an epithelial-mesenchymal transition through Slug induction. These data indicate that NDRG2 suppresses tumor progression via regulating MMP19/Slug axis in gallbladder carcinoma. The loss of NDRG2 is a novel prognostic factor suggesting it could be a useful biomarker in patients with gallbladder carcinoma and a potential therapeutic target.

Citation Format: Nayoung Kim, Jeong-Ki Min, Jin-Man Kim, Hyo Jin Lee. NDRG2 suppresses growth and invasion of gallbladder carcinoma via regulation of matrix metalloproteinase 19/slug interaction. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3404. doi:10.1158/1538-7445.AM2015-3404
Abstract 3407: FGFR4 Arg388 polymorphism is a poor prognostic factor for resected stage III colon cancer
Authors: Cho Sang-Hee , Shin Min-Ho , Kweon Sun-Seog , Kim Hee-Nam , Shim Hyun-Jeong , Hwang Jun-Eul , Bae Woo-Kyun , Chung Ik-Joo
Published: August 2015

Purpose To evaluate the prognostic role of fibroblast growth factor receptor 4 (FGFR4) polymorphism in patients with surgically resected stage III colon cancer.

Patients and Methods FGFR4 polymorphism was checked in peripheral blood from 273 patients who received curative resection and diagnosed as stage III colon cancer. Genomic DNA was extracted from blood which was drawn before curative resection. FGFR4 Gly388Arg genotyping was carried out using high-resolution melting (HRM) analysis.

Results In total 273 patients, 92 patients (34%) were homozygous for Gly388 allele, with 146 heterozygous (54%) and 35 homozygous (13%) for Arg388. No significant association was found between clinical or histopathologic tumor characteristics and FGFR4 genotype. In survival analysis, Gly388 allele showed better 3Y-DFS than Arg388 carriers (heterozygous or Arg388 homozygous) in patients with no adjuvant (p = 0.26) or 5-FU/LV treated (p = 0.038). However, patients receiving adjuvant FOLFOX showed similar 3Y-DFS regardless of genotype. In addition, 5Y-OS was found significant improvement in Gly388 allele compared with Arg388 carriers (92.5% vs. 74.9%, p = 0.04) regardless of adjuvant treatment. In univariate analysis, age, N stage, tumor differentiation, lymphocyte response, adjuvant chemotherapy and FGFR4 genotype were associated with poor OS. In multivariate analysis, FGFR4 Arg388 carriers (HR 5.17; 95% CI, 2.06-12.92, p<0.001) showed significantly poor prognostic factors for OS together with age (≥ 70Y), N stage, poorly differentiation, absence of lymphocyte response, no adjuvant chemotherapy.

Conclusion FGFR4Arg388 polymorphism is an independent poor prognostic factor in surgically resected stage III colon cancer. This result could guide the individual selection for adjuvant chemotherapy and potential therapeutic target for surgically resected stage III colon cancer.

Citation Format: Sang-Hee Cho, Min-Ho Shin, Sun-Seog Kweon, Hee-Nam Kim, Hyun-Jeong Shim, Jun-Eul Hwang, Woo-Kyun Bae, Ik-Joo Chung. FGFR4 Arg388 polymorphism is a poor prognostic factor for resected stage III colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3407. doi:10.1158/1538-7445.AM2015-3407