bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4: Hyper activation of poly(ADP-ribose) polymerase 1 initiates large-scale metabolic changes in a cellular model of glioblastoma
Authors: Wilk Anna M. , Fouquerel Elise , Johnston Bobbie , Trammell Samuel A.J. , Schambeau Lindsay , Andrews Joel F. , Pannell Lewis , Cooper Sara J. , Brenner Charles , Sobol Robert W.
Published: July 2016

PARP1 is a key enzyme of the Base Excision Repair (BER) pathway, facilitating the repair of base damage and single-strand DNA breaks. Activated PARP1 synthesizes poly (ADP-ribose) (PAR), triggering chromatin de-condensation to facilitate recruitment of BER proteins to complete repair. PARP1 activation is attenuated upon successful repair of the DNA lesion. However, unrepaired DNA breaks lead to continuous PARP1 activation and cell death. The molecular mechanism underlying PARP1 activation induced cell death was recently revealed as independent from NAD+ depletion. We have shown that PARP1 activation and PAR synthesis affect glycolysis by directly inhibiting the glycolytic enzyme, hexokinase 1 (HK1). Following on these discoveries, we decided to investigate global metabolic changes triggered by hyperactivation of PARP1. For this study, we used gas chromatography mass spectrometry (GC-MS) to quantify over 150 cellular metabolites and Multiple-Reaction Monitoring Liquid Chromatography Mass Spectrometry (MRM LC-MS) to measure NAD+ metabolites. As a model, we tested glioblastoma cells overexpressing methylpurine DNA glycosylase (MPG) to enhance the PARP1-activation response to DNA damage induced by the alkylating agent MNNG. Simultaneously, to monitor independence from the DNA damaging effect of NAD+ depletion, we utilized an inhibitor of NAD+ biosynthesis, FK866. We found that PARP1 activation leads to a strong accumulation of glucose, likely as a secondary effect of HK1 inhibition. In addition, we observed a significant change in the level of other metabolites including an increase in inosine, inosine monophosphate (IMP), cytidine and uridine levels upon PARP1 activation, suggesting an indirect effect of PARP1 activation on purine and pyrimidine metabolism. Ongoing studies will use these global approaches to unravel the complete metabolic response of cancer cells to genotoxic treatment.

Citation Format: Anna M. Wilk, Elise Fouquerel, Bobbie Johnston, Samuel A.J. Trammell, Lindsay Schambeau, Joel F. Andrews, Lewis Pannell, Sara J. Cooper, Charles Brenner, Robert W. Sobol. Hyper activation of poly(ADP-ribose) polymerase 1 initiates large-scale metabolic changes in a cellular model of glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4.
Abstract 4007: A novel immunotherapy, INT230-6, is able to induce high rates of complete response in mice through a cytotoxic T-cell-dependent mechanism
Authors: Walters Ian B. , Bloom Anja C. , Bender Lewis H. , Terabe Masaki , Berzofsky Jay A.
Published: July 2016

INT-230-6 is a combination of cisplatin, vinblastine and an amphiphilic cell penetration excipient that when administered intratumorally (IT) can induce complete regression in drug-injected and bystander established colon26 tumors. INT230-6 also has the ability to induce long term protection in mice against intravenous (IV) or subcutaneous (SC) re-inoculation of the cancer cells. This long term protection is dependent on induction of both CD4 and CD8 cells as evidenced by in vivo depletion prior to tumor re-inoculation. Previous studies had not elucidated whether the initial drug-injected tumor regression in naive mice was mainly due to the formulation's direct cytotoxicity or whether early tumor regression is dependent also on immune- based cell killing. To address this question, BALB/c mice were inoculated with 1×106 Colon26 cells SC. Tumors were grown to a mean of 300mm3. INT230-6 was administered intratumorally daily for 5 days (day 0 to 4). At the same time mice were treated either with an IgG control, or with rat anti mouse anti-CD4, anti-CD8 or both anti -CD4/CD8 on days 0,1,5,8,15. T cell subset depletion was confirmed by flow cytometry. In preliminary data, INT230-6 treatment resulted in regression from baseline in all mice with a complete regression of the large tumors in 40-80% of mice in the INT230-6 arm alone or the arm with IgG control. The CD4 depleted mice had a similar CR rate of 70%. However, in the CD8 depleted or the dual depleted (CD4/8) mice, tumors regrew after initial regression and no CR was observed. These results suggest that intratumoral administration of INT230-6 in addition to improving the direct cytotoxicity of cisplatin and vinblastine (data shown previously), also induces a potent CD8 tumor-specific T-cell response which participates in regression of the injected lesion, and may be able to similarly target distant lesions such as metastases.

Citation Format: Ian B. Walters, Anja C. Bloom, Lewis H. Bender, Masaki Terabe, Jay A. Berzofsky. A novel immunotherapy, INT230-6, is able to induce high rates of complete response in mice through a cytotoxic T-cell-dependent mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4007.
Abstract 4012: Improving radiotherapy abscopal effects with anti-PD1 and anti-CD137-based immunotherapy
Authors: Rodríguez-Ruiz MariaE. , Rodriguez Inmaculada , Garasa Saray , Barbes Benigno , Solorzano Jose Luis , Perez Gracia Jose Luis , Labiano Sara , Azpilikueta Arantza , Bolanos Elixabet , R. Sanchez-Paulete Alfonso , Aznar M. Angela , Rouzaut Ana , Jure-Kunkel Maria , Etxeberria Iñaki , Alfaro Carlos , Oñate Carmen , Ponz Mariano , Melero Ignacio
Published: July 2016

Radiotherapy is considered an efficacious local tool to erradicate or at least control cancer progression. However, recent lines of preclinical and clinical evidence indicate that proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory monoclonal antibodies (mAb) to act both on irradiated tumor lessions and on distant, non-irradiated tumor sites. The combination of radiotherapy with immunostimulatory anti-PD1 and anti-CD137 mAbs was conducive to favourable effects on distant non-irradiated tumor lesions as observed on transplanted MC38 (colorectal cancer), B16OVA (melanoma) and 4T1 (breast cancer) models. Immunotherapy and radiotherapy synergized both when irradiation was given using external beams or provided with brachytherapy. The therapeutic activity was crucially performed by CD8 T cells, as found in selective depletion experiments. The irradiation regimen induced immune infiltrate changes in the irradiated and non-irradiated lesions featured by reductions in the content of effector T cells, Tregs, and myeloid-derived supresor cells (MDSC), while effector T cells were expressing more intracellular IFN gamma in both the irradiated and contralateral tumors. Importantly, 48h following irradiation CD8+ TILs showed brighter expression of CD137 and PD-1 thereby displaying more target molecules for the activity of the corresponding monoclonal antibodies. Likewise, PD-1 and CD137 were induced on tumor infiltrating lymphocytes from surgically excised of human carcinoma lessions that were irradiated ex-vivo. These findings advocate for clinical development of immunotherapy combinations with anti-PD1 plus anti-CD137 mAbs that can be synergistically accompained by radiotherapy strategies on treatable lesions, even if leaving disease outside the irradiation field.

Citation Format: MariaE. Rodríguez-Ruiz, Inmaculada Rodriguez, Saray Garasa, Benigno Barbes, Jose Luis Solorzano, Jose Luis Perez Gracia, Sara Labiano, Arantza Azpilikueta, Elixabet Bolanos, Alfonso R. Sanchez-Paulete, M. Angela Aznar, Ana Rouzaut, Maria Jure-Kunkel, Iñaki Etxeberria, Carlos Alfaro, Carmen Oñate, Mariano Ponz, Ignacio Melero. Improving radiotherapy abscopal effects with anti-PD1 and anti-CD137-based immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4012.
Abstract 4016: Personalizing the synergy of focal radiation and immunotherapy
Authors: Poleszczuk Jan , Luddy Kimberly , Pilon-Thomas Shari , Schoenfeld Jonathan , Enderling Heiko
Published: July 2016

Tumors grow within a host tissue that both facilitates progression by supplying nutrients and growth factors, and inhibits it through physical constraints and immune surveillance. Transformed cancer cells are confronted with this innate and adaptive immune surveillance, and tumors that develop to become clinically apparent have evolved to evade the immune system. This complex ecological system may be tipped back in favor of effective immune surveillance and tumor regression through novel immunotherapeutic strategies, particularly when used in concert with focal cytotoxic agents such as radiotherapy.

We developed a multi-scale mathematical model framework to simulate local tumor-immune interactions as well as systemic distribution patterns of T cells activated locally in each metastatic site. We simulate focal radiotherapy-induced cytotoxicity and subsequent activation of an adaptive immune response, which may propagate systemically to cause regression of unirradiated metastases, known as the abscopal effect. We simulate concurrent anti-CTLA4 immunotherapy to investigate synergy.

Model simulations suggest that systemic distribution of locally activated T cells is dependent on (i) the anatomical distribution of metastatic sites, (ii) the tumor volume of each metastasis, (iii) the site of focal therapy, (iv) the local therapy protocol of immune activation, and (v) the timing of concurrent immunotherapy. We find that irradiation of different metastatic sites has different likelihoods of inducing systemic tumor regressions.

The developed framework can identify combination therapy protocols that optimally harness the synergy of focal therapy-induced immunity with immunotherapy, and identify optimum treatment targets on a per patient basis.

Citation Format: Jan Poleszczuk, Kimberly Luddy, Shari Pilon-Thomas, Jonathan Schoenfeld, Heiko Enderling. Personalizing the synergy of focal radiation and immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4016.
Abstract 4035: High throughput validation of antibodies for cancer research
Authors: Shulewitz Mark , Hamilton Tim , Schwartz Kaiti , Thornton Christine , Uy Gerald , Archarya Poulomi , Bogoev Roumen , Wang Yan
Published: July 2016

Antibodies are critical reagents both to study and treat a wide range of cancers. In addition to the relevance of antibodies in immunotherapy, their use in understanding cancer pathophysiology is one of the fastest growing applications. Such applications create a constant demand for high quality antibodies that can recognize a wide range of expression of a specific target that is involved in cancer and metastasis. Even though there are thousands of antibodies available, including those from commercial sources, only a small percentage is well characterized and demonstrated to be of dependable quality.

Establishing the ground rules for rigorous validation of antibodies used for immunodetection of cancer targets is critical for obtaining high quality data. In fact, a number of high profile preclinical study failures have been attributed to the use of inadequately characterized antibodies exhibiting non-specific binding or high background. While researchers typically rely on publications in peer-reviewed journals as absolute indicators of antibody quality, this approach has not proven to be very reliable.

In this poster we describe a robust and high throughput process for validating antibodies for use in western blot applications for cancer research. Some key cancer-related target proteins are detected by antibodies and the validation process is illustrated using these western blot data. In a nutshell, each antibody is screened against an entire panel of lysates from different cells expressing endogenous levels of the target protein. A rapid membrane transfer system coupled with a novel stain-free gel imaging technology is used to ensure a high efficiency of transfer and assess protein quality. Antibody sensitivity and binding specificity is determined based on quantitative data generated using an image analysis software. This software allows us to calculate the ratio of specific to non-specific binding accounting for background within the same lane from the western blot image. We have developed analytical parameters that, when applied to these data, allow us to identify high performing antibodies. The basic principles of this validation method are widely applicable for different types of antibodies and sample types. Overall, this approach ensures the selection of high-performing antibody reagents, thus enabling scientists to get dependable data for academic or preclinical research which, in turn, could significantly advance our understanding of cancer and help develop novel immunotherapies for cancer treatment.

Citation Format: Mark Shulewitz, Tim Hamilton, Kaiti Schwartz, Christine Thornton, Gerald Uy, Poulomi Archarya, Roumen Bogoev, Yan Wang. High throughput validation of antibodies for cancer research. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4035.
Abstract 4047: Salmonella protein AvrA activates the STAT3 signaling pathway in colon cancer
Authors: Lu Rong , Wu Shaoping , Zhang Yong-Guo , Xia Yinglin , Zhou Zhongren , Tako Ikuko , Dong Hui , Bissonnette Marc , Sun Jun
Published: July 2016

Salmonella infection in humans can become chronic which leads to low grade persistent inflammation. These chronic infections increase the risk of several gastrointestinal diseases, including cancer. A recent study has shown that antibody against Salmonella flagellin was higher in colorectal cancer and pre-cancer cases than controls in two distinct populations in US and the Netherlands and that dietary intake is the one of the mediating factors, suggesting a potential link of Salmonella to colorectal cancer. Salmonella AvrA is a multifunctional protein that influences eukaryotic cell pathways by regulating ubiquitination and acetylation. In an animal model, we have demonstrated that infection with AvrA expressing Salmonella induces beta-catenin signals and enhances colonic tumorigenesis. Beta-catenin signaling is a key player in intestinal proliferation and tumorigenesis. The relative contributions of AvrA-induced proliferation and inflammation on tumorigenesis, however, are unknown. STAT3 is activated in chronically inflamed intestines in human inflammatory bowel diseases and in colitis-associated colon cancer. In the current study, mice were colonized with Salmonella AvrA-sufficient or AvrA-deficient bacterial strains. Then, inflammation-associated colon cancer was induced through the use of azoxymethane/dextran sulfate sodium. We determined that AvrA expressing bacteria activated the STAT3 pathway, which is predicted to increase inflammation, enhance proliferation, and promote tumorigenesis. Transcriptional activity of STAT3 and its target genes were up-regulated by Salmonella expressing AvrA, thus promoting proliferation and intestinal tumorigenesis. Our current findings provide new insights regarding a STAT3-dependent mechanism by which the specific bacterial product AvrA enhances the development of infection-associated colon cancer. Our observations also raise a note of caution regarding the use of mutant Salmonella organisms as vectors for ant-cancer therapy. Finally, these studies could suggest biomarkers (such as AvrA level in gut) to assess cancer risk in susceptible individuals and infection-related dysregulation of STAT/β-catenin signaling in colon cancer.

Citation Format: Rong Lu, Shaoping Wu, Yong-Guo Zhang, Yinglin Xia, Zhongren Zhou, Ikuko Tako, Hui Dong, Marc Bissonnette, Jun Sun. Salmonella protein AvrA activates the STAT3 signaling pathway in colon cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4047.
Abstract 4050: Bovine leukemia virus small noncoding RNAs are functional elements that regulate replication and contribute to oncogenesis in vivo
Authors: Gillet Nicolas , Hamaidia Malik , de Brogniez Alix , Gutierrez Geronimo , Renotte Nathalie , Reichert Michal , Trono Karina , Willems Luc
Published: July 2016

Bovine leukemia virus (BLV), a retrovirus closely related to the human T-lymphotropic virus type 1 (HTLV-1), induces lymphocytosis and B-cell leukemia/lymphoma in ruminants. Although the provirus undergoes transcriptional silencing during transformation, BLV-infected tumor cells massively express viral miRNAs from internal Pol III promoters. To understand the mechanisms involved, a provirus isogenic to a wild type molecular clone but devoid of miRNAs was inoculated into calves. High-throughput RNA-sequencing and bioinformatic analyzes indicated that BLV miRNAs modulate expression of genes involved in B-cell receptor signaling, apoptosis, cell activation and immune response (GZMA, FOS, PPT1, ANXA1, MAP2K1 and PIK3CG). Direct and indirect interactions within these regulatory pathways were validated by luciferase reporter assays. In vivo, the deletion of BLV miRNAs reduced proviral loads in the natural host, indicating their biological relevance in viral replication and persistence. Furthermore, oncogenesis was abolished in the ovine experimental model. These observations thus show that BLV miRNAs affect key signaling pathways, promote viral replication and drive oncogenesis.

Citation Format: Nicolas Gillet, Malik Hamaidia, Alix de Brogniez, Geronimo Gutierrez, Nathalie Renotte, Michal Reichert, Karina Trono, Luc Willems. Bovine leukemia virus small noncoding RNAs are functional elements that regulate replication and contribute to oncogenesis in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4050.
Abstract 407: Functional analysis of discoidin domain receptor 2 mutation and expression in squamous cell lung cancer
Authors: Kobayashi-Watanabe Naomi
Published: July 2016

The treatment strategy for lung cancer has been rapidly progressed, especially in molecular targeted therapy. However, most treatments are established for adenocarcinoma of lung, but not squamous cell carcinoma (SQCC). SQCC occupies approximately 30% among entire lung cancer patients, but the prognosis is worse than that of adenocarcinoma patients. Discoidin domain receptor (DDR) 2 mutations have been recently reported as one of the candidates of molecular targets for lung SQCC, and they are associated with sensitivity to dasatinib, which is already applicable to treatment of chronic myeloid leukemia. However, precise roles of DDR2 on SQCCs of lung have not been clarified. We performed Sanger sequencing of DDR2 gene in a set of 44 primary lung SQCC samples and 7 lung SQCC cell lines, and identified a novel mutation (T681I) in a lung SQCC patient and a lung SQCC cell line, EBC-1. Endogenous protein expression level of DDR2 measured by Western blot technique was high in a lung SQCC cell line, PC-1. Overexpression of wild type and T681I of DDR2 promoted cancer cell invasion of a human lung SQCC cell line, H226B. In animal model, lung metastasis was enhanced and survival was shortened after transplantation of DDR2 wild type overexpressing cells into NOD/SCID/Jak3null (NOJ) mice, which exhibit deficiencies in NK cell activity, macrophage and dendritic cell function, and complement activation, as well as T and B cell deficiencies. Using the cDNA microarray and protein phosphorylation array assay, we found that ectopic expression of DDR2 induced TGF-β1 protein and mRNA expression accompanied with phosphorylation of c-Jun, p38 MAP kinase, and MMP1 elevation. During the process of metastasis, TGF-β pathway plays pivotal roles especially on disruption of basement membrane through induction of MMPs from fibroblasts mediated through both Smad and non-Smad pathways. Since phosphorylations of c-Jun and p38 MAPK were reported to be involved in non-Smad pathway, DDR2 would induce TGF-β mainly through non-Smad pathway, resulting in cancer invasion and metastasis. Taken together, elevation of DDR2 protein might contribute on tumor progression as well as some of DDR2 mutations in lung SQCC.

Citation Format: Naomi Kobayashi-Watanabe. Functional analysis of discoidin domain receptor 2 mutation and expression in squamous cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 407.
Abstract 4070: Carcinogen 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis is accelerated in Smad3 heterozygous mice compared to Smad3 wild type mice
Authors: Liu Zhengxue , Kundu Tanima R. , Matsuura Isao , Wang Guannan , Lin Yong , Lou You-Rong , Barnard Nicola J. , Wang Xiao-Fan , Huang Mou-Tuan , Suh Nanjoo , Liu Fang
Published: July 2016

Smad3 plays an important role in inhibiting cell proliferation and promoting apoptosis. Previous studies based on cell culture and xenograft animal models suggest that Smad3 has tumor suppressor function for breast cancer during early stages of tumorigenesis. Here we show that DMBA (7,12-dimethylbenz[a]anthracene), a chemical carcinogen, induces mammary tumor formation at a significantly higher frequency in the Smad3 heterozygous mice than in the Smad3 wild type mice. This is the first genetic evidence showing that Smad3 inhibits mammary tumorigenesis in a mouse model. Our findings provide strong support that Smad3 has important tumor suppressor function for breast cancer.

Citation Format: Zhengxue Liu, Tanima R. Kundu, Isao Matsuura, Guannan Wang, Yong Lin, You-Rong Lou, Nicola J. Barnard, Xiao-Fan Wang, Mou-Tuan Huang, Nanjoo Suh, Fang Liu. Carcinogen 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis is accelerated in Smad3 heterozygous mice compared to Smad3 wild type mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4070.
Abstract 4081: Stromal autophagy is required for the growth of cutaneous melanoma
Authors: Zhang Ran Ellen , Guo Natalie J. , Krishnan Nithya , Jelinek David , Uzunbas Gizem Karsli , Xie Xiaoqi , Gertych Arek , Mehnert Janice M. , Knudsen Beatrice , White Eileen , Coller Hilary A.
Published: July 2016

Through degrading bulk cytoplasmic materials into intermediate building blocks, autophagy refuels metabolism and maintains homeostasis under starvation and stress conditions. The context-dependent role of autophagy in cancer cells during tumorigenesis and tumor progression has been explored over the past decade; however, studies of autophagy in relation to the tumor microenvironment are still lacking. In this study, we investigated the role of autophagy in the cancer-associated fibroblasts (CAFs), a major component of the microenvironment that is important for the growth and development of cutaneous melanoma. Analysis of clinical biopsies revealed high levels of autophagy in fibroblasts that surrounded melanoma cells, but not in the non-tumor adjacent area. Fibroblasts co-cultured with any of multiple melanoma cell lines, but not melanocytes, also showed increased autophagic flux, which requires transforming growth factor beta (TGF-β) signaling. Knocking down autophagy-related (ATG) genes ATG5 or ATG7 in fibroblasts did not impair their proliferation in vitro, but inhibited the growth of co-engrafted melanoma cells in vivo. Further, by introducing intradermal melanoma allografts into mice that harbored fibroblast-specific Atg7 deletion, we observed remarkable tumor growth retardation. In order to mimic anti-autophagy therapy for patients, we generated mice that allowed conditional Atg7 depletion in their entire bodies, and found significant tumor growth suppression. Atg7 knockout mice contained elevated plasma levels of immune cell attractants, including CXCL9, CXCL10 and MCP-1. By inhibiting these cytokines/chemokines with atorvastatin, tumor growth in Atg7 knockout mice was completely rescued; while, there was no effect of atorvastatin on control mice. Taken together, our data show that through modulating the tumor microenvironment, stromal autophagy plays an important role during melanoma growth and development. Our findings indicate that stromal autophagy may represent a valuable therapeutic target that might induce less side effects and systemic toxicity.

Citation Format: Ran Ellen Zhang, Natalie J. Guo, Nithya Krishnan, David Jelinek, Gizem Karsli Uzunbas, Xiaoqi Xie, Arek Gertych, Janice M. Mehnert, Beatrice Knudsen, Eileen White, Hilary A. Coller. Stromal autophagy is required for the growth of cutaneous melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4081.
Abstract 4097: Significance of glypican-1 expression on tumor stromal cells in gastric carcinoma
Authors: Miki Yuichiro , Yashiro Masakazu , Kitayama Kishu , Kasashima Hiroaki , Masuda Go , Kubo Naoshi , Sakurai Katsunobu , Toyokawa Takahiro , Tanaka Hiroaki , Muguruma Kazuya , Ohira Masaichi , Hirakawa Kosei
Published: July 2016

Background: Glypican1 (GPC1) is a cell surface heparan sulfate proteoglycan that acts as a co-receptor for heparin-binding growth factors, and it plays a crucial role in cancer growth. Recently, it has been reported that GPC1 enriched on cancer-derived exosomes, and GPC1-positive exosomes in the circulation was frequently found in the patients with early pancreatic cancer. However role of GPC1-positive exosomes derived from tumor stromal cells remains to be unclear. The aim of this study is to clarify the significance of GPC1 expression on stromal cells in gastric carcinoma.

Methods: A total of 597 patients with gastric cancer was enrolled in this study. An anti-GPC1 antibody was used for immunohistochemical staining. GPC1 expression was scored by the intensity of staining and the percentage of positive cells. GPC1 expression on cancer cells or stromal cells was evaluated. The corelation between GPC1 expression and clinicopathological features was statistically analyzed.

Results: GPC1 expression on cancer cells was found in 473 (79%) of 597 cases. GPC1 expression on stromal cells was found in 147 (75%) of 597 cases. A Kaplan-Meier survival curve showed that overall survival of patients with GPC1-positive stromal cells was significantly better (log-rank p = 0.0028) than that with GPC1-negative stromal cells (5-year survival rate; 72.5% vs 57.9%, respectively). In contrast, overall survival was not statistically different (log rank p = 0.090) between patients with GPC1-positive tumor cells and GPC1-negative tumor cells (5-year survival rate; 71.8% vs. 64.6%, respectively). GPC1 expression on stromal cells was significantly correlated with T stage, lymph node metastasis, lymphatic vessel invasion, and clinical stage.

Conclusions: GPC1 expression on stromal cells was associated with the good prognosis of patients with gastric cancer. GPC1-positive exosomes in stromal cells might be correlated with the progression of gastric cancer.

Citation Format: Yuichiro Miki, Masakazu Yashiro, Kishu Kitayama, Hiroaki Kasashima, Go Masuda, Naoshi Kubo, Katsunobu Sakurai, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Hirakawa. Significance of glypican-1 expression on tumor stromal cells in gastric carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4097.
Abstract 410: Exhaustive scanning of mutation hotspot regions using a new tool: The MHS-ddPCR
Authors: Decraene Charles , Bidard Francois-Clement , Belaabi Samia , Rouleau Etienne , Saliou Adrien , Houy Alexandre , Milder Maud , Lantz Olivier , Ychou Marc , Pierga Jean-Yves , Stern Marc-Henri , Proudhon Charlotte
Published: July 2016

Background: Recent progress in the ‘liquid biopsy’ field in combination with the development of the droplet digital PCR (ddPCR) technology now enables non-invasive monitoring of cancer-related genomic alterations with high detection accuracy. Current ddPCR techniques represent the gold standard for the detection of point mutations that have been previously characterized on tumor tissues. However, ddPCR screen for only one mutation (or a few mutations, with multicolor ddPCR) per reaction. This is a clear limitation to use it as a discovery tool, to detect resistance-associated mutations that may appear during therapy (e.g. ESR1 activating mutations in breast cancer) or when tumor tissue is not available (e.g. EGFR activating mutations in lung cancers).

Methods:

We developed the Multiple Hotspot mutations detection by Single droplet digital PCR (MHS-ddPCR) method, a variant of the conventional ddPCR technique, which detects all genomic alterations within a hotspot region, using a unique couple of Taqman oligo-probes. We first established two specific assays covering KRAS and EGFR mutation hotspot regions, which are of clinical importance in the context of colorectal cancer. The assay for KRAS scans for the 7 most common mutations in codons 12 and 13 of the gene as well as all other mutations with lower frequency (<1%). The EGFR assay screens for all in-frame deletions of exon 19, which are frequent activating events in EGFR.

Results:

Sensitivity and specificity of MHS-ddPCR were assessed on decreasing fractions of tumor DNA mutated for KRAS. The KRAS assay reaches a sensitivity of 0.02% and was validated on plasma and tumor samples harboring a panel of different KRAS mutations. Similarly, the EGFR assay can detect numerous exon 19 deletions from patient samples and with limited amounts of tumor DNA.

Conclusions:

The MHS-ddPCR is extremely sensitive, works for numerous hotspot regions with different types of alterations (SNV, deletion) and is cost-effective as it combines multiple assays in one reaction.

Citation Format: Charles Decraene, Francois-Clement Bidard, Samia Belaabi, Etienne Rouleau, Adrien Saliou, Alexandre Houy, Maud Milder, Olivier Lantz, Marc Ychou, Jean-Yves Pierga, Marc-Henri Stern, Charlotte Proudhon. Exhaustive scanning of mutation hotspot regions using a new tool: The MHS-ddPCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 410.
Abstract 4122: Ceramide is a key factor that regulates the crosstalk between TGF-ß and sonic hedgehog signaling at the basal cilia to control cell migration and tumor metastasis
Authors: Gencer Salih , Oleinik Natalia , Dany Mohammed , Ogretmen Besim
Published: July 2016

Recent studies indicate that ceramide species with different fatty-acid chain lengths play diverse biological functions in various cellular processes, highlighting the importance of ceramide synthases (CerS) in these processes. Migration and cell mobility, a part of these processes, also are effected by ceramide metabolism. However, the molecular mechanism of CerS/and ceramide involved is unknown. Here, we investigated the effect of CerS/and ceramide on migration and its related signal pathways in situ and in vivo model. Interestingly, our data show that among CerS only CerS4/ceramide is involved to cell migration and tumor metastasis. Here, we also have generated CerS4-/- mice for in vivo studies. Interestingly, we observed that genetically loss of CerS4 resulted in severe irreversible alopecia, which was associated with hyper-proliferation and migration of keratinocytes. Mechanistically, we show here that genetic loss or shRNA-mediated knockdown of CerS4 enhances cell migration by which ligand-independent signaling of TGF-beta receptors I and II in various cell types, including keratinocytes, mouse embryonic fibroblasts and cancer cells. Moreover, we found that ceramide directly interact with Smad7 and this interaction was decreased by shRNA-mediated knockdown of CerS4. Thus, ceramide-Smad7 binding modulates plasma membrane association of TGF-ßR1 at primary basal cilia, and inhibits its signaling through Sonic-Hedgehog (Shh) for migration. Furthermore, Ceramide accumulation at the primary basal cilia was decreased by knockdown of CerS4. Thus, these data revealed that CerS4/ceramide signaling plays key roles in the regulation of cell migration and metastasis via controlling the TGF-ßR and Shh axis at primary basal cilia.

Citation Format: Salih Gencer, Natalia Oleinik, Mohammed Dany, Besim Ogretmen. Ceramide is a key factor that regulates the crosstalk between TGF-ß and sonic hedgehog signaling at the basal cilia to control cell migration and tumor metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4122.
Abstract 4132: Multiple drug resistance-associated protein 4 (MRP4): Role in triple negative breast cancer
Authors: Kochel Tyler , Ma Xinrong , Kundu Namita , Reader Jocelyn , Goloubeva Olga , Fulton Amy M.
Published: July 2016

Introduction: MRP4 is responsible for the active export of prostaglandin E2 (PGE2) from cells. PGE2, derived from cyclooxygenase-2-mediated synthesis, contributes to tumor growth and metastasis of many epithelial tumors; however, the role of MRP4 in breast cancer has not been investigated. Methods: We employed publicly available breast cancer gene expression datasets and a panel of breast cancer cell lines to examine the expression and function of MRP4 as well as other members of the PGE2 pathway. In contrast to the export function of MRP4, the prostaglandin transporter PGT/SLCO2A1 imports PGE2 into the cell where 15-prostaglandin dehydrogenase (5-PGDH/HPGD) metabolizes PGE2 to terminate ligand-mediated activation of the prostaglandin E (EP) receptors. Results: We found that ABCC4, which encodes the MRP4 protein, is widely expressed in breast cancer but breast cancer subtypes have different expression profiles of PGE2 pathway members. ABCC4/MRP4 is elevated more frequently in basal breast tumors than in apocrine or luminal tumors. This is the first report that elevated expression of MRP4 is found in breast cancer subtypes (basal, HER2-enriched) with the worst prognoses. ABCC4 expression was also higher in breast cancer-associated stroma compared to healthy mammary tissue. Basal-type tumors also expressed the highest levels of PTGS2/COX-2 while the genes responsible for PGE2 metabolism (HPGD and SLCO2A1) are negatively correlated with TNBC. The same pattern was observed in breast cancer cell lines, where basal-type and HER2-enriched cell lines highly express MRP4 but luminal breast cancers do not. HPGD was decreased in most cancer cell lines compared to MCF10A immortalized cells, consistent with the known tumor suppressor role of HPGD/15PGDH. MRP4 exports PGE2 which was reduced in the presence of MRP4 small m.w. inhibitors or by MRP4 gene silencing. Lastly, expression of shMRP4 in highly metastatic, basal-type MDA-MB-231 human breast cancer cells reduced the ability of these cells to spontaneously metastasize to the lungs of NOD/SCID mice but did not affect the tumor growth properties of tumor cells implanted proximal to the mammary gland. Conclusions: Elevated MRP4 is a characteristic of aggressive breast cancer subtypes and is partially responsible for the high levels of PGE2 in the tumor microenvironment. Silencing of MRP4 reduces spontaneous metastasis in a xenograft model of TNBC. Further investigation of MRP4 as a potential therapeutic target is warranted.

Citation Format: Tyler Kochel, Xinrong Ma, Namita Kundu, Jocelyn Reader, Olga Goloubeva, Amy M. Fulton. Multiple drug resistance-associated protein 4 (MRP4): Role in triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4132.
Abstract 4151: APC control of retinoic acid suppresses immune cell mediated intestinal proliferative response
Authors: Templeton Amanda K. , Hammoud Saher , Cairns Brad , Jones David
Published: July 2016

Adenomatous polyposis coli (APC) tumor suppressor function is centered on its role in antagonizing Wnt/B-catenin mediated colonic proliferation. Our previous studies, however, established a second role wherein APC's control of retinoic acid (RA) results in intestinal epithelial cell differentiation. Surprisingly, the loss of APC or RA alone in zebrafish embryos was not sufficient to drive nuclear accumulation of B-catenin or intestinal cell proliferation. However, combining APC or RA deficiency with a KRAS mutation was sufficient to induce nuclear localization of B-catenin and the proliferative response. In contrast, the loss of RA alone in adult zebrafish results in both intestinal differentiation defects and proliferation. We were therefore interested in investigating these seemingly contradictory results. Using adult zebrafish, we show for the first time that following loss of retinoic acid adaptive immune cells (but not innate immune cells) are indirectly required to elicit the proliferative response independent of Kras activation. We demonstrate that IL-17 secretion from adaptive immune cells in response to loss of RA predisposes colonic epithelium to inflammation resulting in compromised barrier function, loss of cell polarity, reactivation of a progenitor intestinal program, and proliferation. Importantly, we find in both mouse and human intestinal organoid cultures an evolutionarily shared response with zebrafish of T cell dependent intestinal inflammation and proliferation following loss of RA. Our model demonstrates that loss of RA causes an increase in inflammatory mediators (Cox2 and Prostaglandins) that induces infiltration of IL-17 secreting T cells. In a feed-forward loop, the IL-17 acts on the primed epithelium to up-regulate proliferative signaling mediators (Raf and Rac1). Together, these studies provide an important new perspective on the ordering of molecular events that may underlie colon tumor progression.

Citation Format: Amanda K. Templeton, Saher Hammoud, Brad Cairns, David Jones. APC control of retinoic acid suppresses immune cell mediated intestinal proliferative response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4151.
Abstract 4195: Targeting the immune microenvironment to improve colorectal cancer anti-angiogenic therapy
Authors: Jung Keehoon , Heishi Takahiro , Yun Andy , Padera Timothy P. , Jain Rakesh K. , Fukumura Dai
Published: July 2016

Colorectal cancer (CRC) is the leading cause of cancer-related death worldwide. Anti-angiogenic therapy with bevacizumab has been proven efficacious in metastatic CRC. However, the improvement in overall survival has been modest and rather disappointing. Thus, there is an urgent need to identify the underlying mechanisms responsible for the resistance to anti-angiogenic therapy and develop strategies to overcome it. Clinical and preclinical interest in tumor immunotherapy has been renewed in a significant manner recently. However, the role of the immune microenvironment in colorectal cancer resistance to anti-angiogenic therapy is not well understood.

We have developed miniaturized confocal endomicroscopy technique for spontaneous CRC models in mice. We also established a novel cecum window recently. These unique systems enable us to monitor the CRC and its immune microenvironment longitudinally with a video-rate intravital multi-photon microscope. Using these CRC models and in vivo imaging methods, we found that anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody increases infiltration of Gr1+ myeloid-derived suppressor cells (MDSCs) and they alter the tumor microenvironment toward an immune-suppressive condition to avoid attack of the host immune system. We also found that the Gr1+ MDSCs express CXCR4, which has important roles in cell-autonomous survival and chemotaxis, through its downstream signaling pathways. When we treat the VEGF signaling blocker together with a CXCR4-specific inhibitor, the level of the Gr1+ MDSC infiltration is dramatically decreased nearly to that of control group, and subsequently, tumor growth is significantly delayed compared to separate treatment of each inhibitor.

Taken together, we identified Gr1+ MDSCs as key players in tumor resistance to anti-angiogenic therapy in CRCs, and the blockade of CXCR4 signaling has a synergistic effect with anti-VEGF therapy, proven by state-of-the-art in vivo imaging modalities.

Citation Format: Keehoon Jung, Takahiro Heishi, Andy Yun, Timothy P. Padera, Rakesh K. Jain, Dai Fukumura. Targeting the immune microenvironment to improve colorectal cancer anti-angiogenic therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4195.
Abstract 4206: A dual-motif CAIX inhibitor, [64Cu]XYIMSR-06, for PET imaging of clear cell renal cell carcinoma
Authors: MINN IL , Koo Soo Min , Lee Hye Soo , Brummet Mary , Rowe Steven P. , Gorin Michael A. , Sysa-Shah Polina , Lewis William D. , Ahn Hye-Hyun , Wang Yuchuan , Ray Sangeeta , Mease Ronnie C. , Nimmagadda Sridhar , Allaf Mohamad E. , Pomper Martin G. , Yang Xing
Published: July 2016

Carbonic anhydrase IX (CAIX) is a cell surface enzyme that is over-expressed in approximately 95% of clear cell renal cell carcinomas (ccRCCs), the most common renal cancer. CAIX is also a surrogate marker of hypoxic tumors for various human cancers; however, a clinically viable molecular imaging agent targeting CAIX is not available. We have synthesized and performed in vitro and in vivo evaluation of a dual-motif, low-molecular-weight inhibitor of CAIX, [64Cu] XYIMSR-06, for imaging CAIX expression on ccRCC tumors using positron emission tomography (PET). [64Cu] XYIMSR-06 was synthesized in two steps from reported dual-motif precursor 1. Upon radiolabeling, [64Cu] XYIMSR-06 was evaluated in immunocompromised mice bearing CAIX-expressing SK-RC-52 tumors for in vivo PET imaging and biodistribution. [64Cu] XYIMSR-06 was generated in radiochemical yields of 51.0 ± 4.5% (n = 5) and specific radioactivity of 6.0 GBq/μmol (170Ci/mmol ± 70, n = 5). Tumor could be visualized on PET images by 1 h post-injection with high tumor-to-background levels achieved within 24 h. Biodistribution studies of [64Cu] XYIMSR-06 demonstrated a maximum tumor uptake of 19.3 ± 4.51% injected dose per gram of radioactivity at 4 h. Tumor-to- blood, muscle and kidney ratios were 129.6 ± 18.8, 84.3 ± 21.0 and 2.1 ± 0.26, respectively, at 8 h post-injection. At 24 h, a tumor-to-kidney ratio of 7.1±2.5 was achieved. These findings represent increased tumor-to-background ratios at the indicated times relative to previously reported CAIX-targeted imaging agents. [64Cu] XYIMSR-06 is a promising candidate for PET imaging of CAIX expressing tumors, especially ccRCC.

Citation Format: IL MINN, Soo Min Koo, Hye Soo Lee, Mary Brummet, Steven P. Rowe, Michael A. Gorin, Polina Sysa-Shah, William D. Lewis, Hye-Hyun Ahn, Yuchuan Wang, Sangeeta Ray, Ronnie C. Mease, Sridhar Nimmagadda, Mohamad E. Allaf, Martin G. Pomper, Xing Yang. A dual-motif CAIX inhibitor, [64Cu]XYIMSR-06, for PET imaging of clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4206.
Abstract 424: Urine cf-DNA SNPs as early biomarkers of prostate cancer
Authors: Datta Amrita , Kim Hogyoung , Feibus Allison , Badoo Melody , Johnson Adedoyin , Moustafa Ahmed , Silberstein Jonathan , Moparty Krishnarao , Thomas Raju , Abdel-Mageed Asim B.
Published: July 2016

Background: The current strategies of prostate cancer (PC) detection, such as serum PSA, digital rectal examination (DRE) and prostate biopsy have been hampered by many limitations, including the degree of aggressiveness, invasiveness, non-specificity to PCa, over diagnosis and over treatment. Recent studies have proposed the use of cell-free circulating DNA (cf-DNA) as non-invasive biomarkers for cancer diagnosis, prediction of tumor burden and prognosis. We hypothesize that a selective subset of PC associated Single Nucleotide Polymorphisms (SNPs) in urine cf-DNA detects PCa with high specificity and sensitivity.

Aim: To identify PC specific SNPs in urine cf-DNA from prostate cancer (PC) patients and further validate their clinical utilities as non-invasive biomarkers for early detection of PC in comparison to other genitourinary conditions, such as benign prostate hyperplasia (BPH) and prostatitis.

Method and Results: A protocol was optimized for collection, processing and storage of urine samples and isolation of urine cf-DNA. Our discovery dataset was constructed by comparative Illumina HiSeq next generation sequencing (NGS) of urine cf-DNA from three different cohorts of patients presenting with PC (Gleason score 6), benign prostatic hyperplasia (BPH) or prostatitis. The data was analyzed by SNAPE-pooled and annotated using ANNOVAR; revealing 24 PC-specific non-synonymous SNPs. The selective detection of urine cf-DNAs in PC patients was verified by conventional PCR using primers flanking their genomic sites. Our validation analysis was performed in patients (n = 50/group) diagnosed with PC (Gleason score ≤ 3+4), Gleason score ≥ than 4 + 3, or patients deemed as “no cancer seen”. This was performed by Droplet Digital PCR (ddPCR) targeting wild-type versus variant (SNP) DNA in each patient sample. The results have revealed detection of two PC specific SNPs in urine cf-DNA (>60% for SNP1 and >75% SNP2 in 91 patients; patent pending).

Conclusion: Our study brings forth the potential clinical utility for two novel urine cf-DNA SNPs as early biomarkers for prostate cancer.

Citation Format: Amrita Datta, Hogyoung Kim, Allison Feibus, Melody Badoo, Adedoyin Johnson, Ahmed Moustafa, Jonathan Silberstein, Krishnarao Moparty, Raju Thomas, Asim B. Abdel-Mageed. Urine cf-DNA SNPs as early biomarkers of prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 424.