bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4117: AMD070, a novel orally bioavailable CXCR4 inhibitor, inhibits the metastases of oral cancer via SDF-1/CXCR4 system
Authors: Kinouchi Makoto , Uchida Daisuke , Kuribayashi Nobuyuki , Wakui Takahiro , Kuribayashi Kyoko , Okubo Maki , Saito Masahiro , Masuyama Eri , Kawamata Hitoshi
Published: July 2016

We have demonstrated that the stromal cell-derived factor (SDF)-1/CXCR4 system is involved in metastatic processes in oral cancer. Moreover, we have also reported that blocking CXCR4 with AMD3100, a CXCR4 antagonist, may be a potent anti-metastatic therapy for CXCR4-related head and neck cancer. Recently, AMD070 has been described as a novel orally bioavailable inhibitor of CXCR4, being minimally invasive than AMD3100. In this study, we examined the effect of AMD070 on the SDF-1/CXCR4 dependent-metastases in oral cancer cells, B88-SDF-1, which express high levels of SDF-1 and CXCR4.

We examined the effect of CXCR4-specific antagonists, AMD3100 and AMD070 on the cell growth, cell migration and cell invasion of B88-SDF-1 cells. Both antagonists did not affect the anchorage-dependent growth of B88-SDF-1 cells, but did inhibit the SDF-1/CXCR4 dependent-migration of the cells. Moreover, AMD070 significantly suppressed anchorage-independent growth and invasion of B88-SDF-1 cells. Next we examined the effect of AMD070 to distant metastasis in vivo. Daily administration of AMD070 per os significantly inhibited the metastasis of B88-SDF-1 cells to the lungs of nude mice. These results indicated that AMD070 might be a novel orally bioavailable inhibitor on the metastases of oral cancer by the SDF-1/CXCR4 system.

Citation Format: Makoto Kinouchi, Daisuke Uchida, Nobuyuki Kuribayashi, Takahiro Wakui, Kyoko Kuribayashi, Maki Okubo, Masahiro Saito, Eri Masuyama, Hitoshi Kawamata. AMD070, a novel orally bioavailable CXCR4 inhibitor, inhibits the metastases of oral cancer via SDF-1/CXCR4 system. [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 4117.
Abstract 412: RNA-Seq provides cost-effective alternative for typing self-identifying antigens in immunotherapy patients
Authors: Robasky Kimberly , Powers Jason , Trapolsi Donald , Jasper Jeff , Brown Chad
Published: July 2016

The excitement surrounding immunotherapy is being driven by results in the clinic. Currently, autoimmunity is an unfortunate side-effect for a large fraction of those treated. Consequently, understanding how self-antigens are recognized is nearly as important as characterizing the immune repertoire for efficacious delivery of these promising new therapies. One approach to measuring the tumor immune environment is with RNA-Seq assays. Here we show that the genes responsible for presenting self-antigens (HLA Class I and Class II) can also be ascertained from RNA-Seq on total RNA. We present the detailed results from standard RNA-Seq pipeline analysis for 40 lymphoblastoid cell lines to achieve 91.3% overall concordance with “gold standard” typings. These samples were sequenced with 50bp paired-ends and approximately 30M reads. We additionally present RNA-Seq analysis results from calling HLA alleles on 15 replicate pairs of FFPE hepatic tumor samples, finding 88.33% overall replicate concordance with 2-digit precision, and 90% for Class I alleles. Finally, we present HLA-types on triplicates from 2 common breast cancer cell lines, MCF7 and T47D for loci HLA-A,-B,-C,-DRB1,-DQB1, with 90% overall replicate concordance. Emerging targeted DNA-Seq assays aimed at high-throughput clinical trials require high quality whole-blood samples that yield larger amounts of assay input material. Alternatively, the analyses presented here do not require HLA-region enrichment and thus can also be performed on legacy RNA-Seq data. Notably, the data here are generated from sample types and amounts that are more typical to a clinical oncology setting, and because it does not rely on targeted capture as do DNA-Seq assays, these analyses hold greater promise for assembling rare alleles and fusions.

Citation Format: Kimberly Robasky, Jason Powers, Donald Trapolsi, Jeff Jasper, Chad Brown. RNA-Seq provides cost-effective alternative for typing self-identifying antigens in immunotherapy patients. [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 412.
Abstract 4124: Sub-cytotoxic concentrations of a novel polyphenol fatty acid ester derivative arrest breast cancer cell proliferation and metastasis
Authors: Fernando Wasundara , Coombs Melanie R. Power , Hoskin David W. , Rupasinghe H. P. Vasantha
Published: July 2016

In this study, the antiproliferative and antimetastatic properties of phloridzin docosahexaenoate (PZ-DHA), a novel polyphenol fatty acid ester derivative, were explored using in vitro and in vivo models of mammary carcinoma. PZ-DHA combines phloridzin (PZ, a dihydrochalcone found in apple peels) with docosahexaenoic acid (DHA, an omega-3 fatty acid). The regioselective acylation reaction was catalyzed by lipase B enzyme from Candida antarctica. PZ-DHA-induced selective cytotoxicity towards breast cancer cells (triple-negative mammary carcinoma cells, MDA-MB-231, MDA-MB-468, and 4T1; estrogen receptor-positive mammary carcinoma cells, MCF-7 and T-47D) was observed compared to human mammary epithelial cells (HMECs and MCF 10A) using the MTS assay. Significantly less (p<0.05) cytotoxicity of PZ-DHA for normal cells was also confirmed using flow cytometric analysis of annexin-V-FLUOS/propidium iodide-stained MCF 10A and human dermal fibroblasts compared to MDA-MB-231 cells. Flow cytometric analysis of Oregon Green 488-stained MDA-MB-231 cells showed an antiproliferative effect of PZ-DHA at sub-cytotoxic concentrations (10 to 30 μM). Cell cycle analysis showed that MDA-MB-231 replication was arrested at the G2/M phase of the cell cycle following treatment with PZ-DHA at sub-cytotoxic concentrations. PZ-DHA suppressed the migration of MDA-MB-231 and 4T1 cells in vitro in wound healing and cell migration assays. Reduced expression of proteins (β-catenin, slug, Zinc finger E-box-binding homeobox 1, vimentin and matrix metalloproteinase-2) involved in the epithelial-to-mesenchymal transition was demonstrated by western blot analysis of PZ-DHA-treated MDA-MB-231 cell lysates. Finally, 4T1 tumor bearing-female BALB/c mice that received intra-peritoneal injections of PZ-DHA showed a significant reduction (p<0.05, n = 9) in primary tumor volume at the mammary fat pad and fewer metastatic lesions in the lungs compared to the saline-treated control mice, suggesting an in vivo antimetastatic effect of PZ-DHA. In conclusion, findings of this study reveal that PZ-DHA suppresses mammary carcinoma cell proliferation and metastasis, suggesting a potential clinical application to prevent breast cancer progression in patients.

Citation Format: Wasundara Fernando, Melanie R. Power Coombs, David W. Hoskin, H. P. Vasantha Rupasinghe. Sub-cytotoxic concentrations of a novel polyphenol fatty acid ester derivative arrest breast cancer cell proliferation and 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 4124.
Abstract 4126: An anti-CD4 antibody protects mice from anti-PD-1/PDL-1 antibody-induced fatal anaphylaxis and shows potent anti-metastatic activity in the 4T1 spontaneous metastasis model
Authors: Ito Satoru , Yokochi Shoji , Ishiwata Yoshiro , Ueha Satoshi , Shand Francis , Matsushima Kouji
Published: July 2016

[Introduction]

We previously reported that an α-CD4 depleting monoclonal antibody (mAb) has very potent anti-tumor effects in several murine tumor models, particularly when combined with α-PD-1/L1 mAbs (Cancer Immunol. Res. 3: 631-640; 2015). Here we examined the effect of this α-CD4 mAb on 4T1 murine mammary tumors, which undergo spontaneous metastasis from an orthotopically transplanted site to the lung.

[Methods]

4T1 tumor cells (1 × 105) were injected into the mammary fat pad of female BALB/c mice and primary tumor volume was measured every 2-3 days. Spontaneous lung metastasis was quantitated by removing the lungs, fixing them in Bouin's fixative, and counting the number of metastatic nodules on the surface of lung.

[Results]

Administration of α-CD4 mAb (clone GK1.5; days 5 and 9 after tumor inoculation) significantly inhibited 4T1 lung metastasis (n = 10) without affecting primary tumor size. α-PD-1 and α-PD-L1 mAbs had minimal effects on primary tumor growth and metastasis. However, administration of α-PD-1 or α-PD-L1 mAbs triggered an IgG1-mediated fatal anaphylactic reaction upon the third or fourth administration. Interestingly, the anaphylactic reaction was completely prevented by co-administration of the α-CD4 mAb.

[Discussion]

Based on these results, α-CD4 mAb may be useful for preventing metastasis associated with advanced cancer. In addition, α-CD4 mAb appeared to be protective against the IgG-mediated anaphylactic reaction and autoimmune-like side effects that are associated with α-PD-1/α-PD-L antibody administration-induced enhanced B-cell immunity. Increased myelopoiesis due to high G-CSF production by 4T1 cells may have contributed to the anaphylactic reaction.

Citation Format: Satoru Ito, Shoji Yokochi, Yoshiro Ishiwata, Satoshi Ueha, Francis Shand, Kouji Matsushima. An anti-CD4 antibody protects mice from anti-PD-1/PDL-1 antibody-induced fatal anaphylaxis and shows potent anti-metastatic activity in the 4T1 spontaneous metastasis model. [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 4126.
Abstract 413: Validation of an antibody independent tool for patient selection: RNA in situ hybridization detects Met expression levels predictive for response to Met inhibition by Bay 853474
Authors: von Ahsen Oliver , Krahn Thomas , Schatz Christoph
Published: July 2016

This study was performed in order to validate RNA in situ hybridization (RNA ISH) as tool for patient selection. Met expression was analyzed in a matched sample set of FFPE versus fresh frozen tumor samples comprising 20 cases of gastric cancer. Classical immunohistochemistry using the antibody SP44 and RNA ISH (RNAscope by ACD) were used to detect c-met expression in FFPE material. The results were confirmed by sandwich-immunoassays on Met and its phosphorylation on tyrosine 1349 (MSD) as well as mass spectrometry. The level of functional relevance was determined by testing a set of cell lines comprising some with genomic amplification of the met gene as well as some non-amplified lines showing different expression levels. Among gastric cancer cell lines only those with met amplification respond to treatment with the small molecule Met inhibitor Bay 853474. The cell line result generates a responder hypothesis that can be used to define a cutoff for clinical samples. 2 of the 20 investigated clinical samples were shown to have high level Met expression by RNA ISH and IHC that could be confirmed by sandwich-immunoassays also showing high level of functional activity by phosphotyrosine 1349. Met expression in these cases was also confirmed by mass spectrometry. Expression levels and functional activity in these 2 cases were in the range that predicts response to treatment as established with gastric cancer cell lines. Determination of predictive biomarkers by immunohistochemistry can be limited due to lack of high quality antibodies of sufficient specificity. Due to its high specificity, RNA in situ hybridization is a technique that can be used to confirm the findings obtained by immunohistochemistry and may potentially even replace immunohistochemistry it if no suitable antibodies are available or not specific enough e.g. to discriminate between closely related protein isoforms. We show the biological relevance of RNA in situ hybridization on FFPE samples by correlation with immunohistochemistry, ELISA based approaches and mass spectrometry. RNA ISH is shown to be specific and sensitive enough to identify cases of functionally relevant MET overexpression levels in gastric cancers and can be used to select patients for treatment.

Citation Format: Oliver von Ahsen, Thomas Krahn, Christoph Schatz. Validation of an antibody independent tool for patient selection: RNA in situ hybridization detects Met expression levels predictive for response to Met inhibition by Bay 853474. [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 413.
Abstract 4130: A novel tumor-suppressor role for squalene epoxidase in human colorectal metastasis
Authors: Jun Soo Young , Cho Hyun-Soo , Lee Jeong-Ju , Yoon Ji-Yong , Ahn Jun-Ho , Min Ju-Sik , Jeon Sujin , Lee Jae-Hye , Choi Min Hyuk , Kim Nam-Soon
Published: July 2016

Malignancy cancer cells are reported to develop mechanisms to resist anoikis (apoptotic cell death from deprivation of cell-cell/matrix interactions), thereby enhancing the survival of cancer cells and secondary tumor formation in distant organs, which is responsible for 90% of cancer-related fatalities. A high serum level of cholesterol (hypercholesterolemia) in patients with colorectal cancers (CRCs) metastasis is reported, but the underlying relationship were never elucidated. Human CRC tissues revealed that the expression of squalene epoxidase (SqE), the rate-limiting enzyme in cholesterol synthesis, is significantly reduced in the advanced stages of CRC. Here we report that the knockdown/degradation of SqE by either the application of small-interfering RNA against SqE or a high-level of cholesterol significantly potentiates CRC cell survival/metastasis via the induction of an epithelial-mesenchymal transition as well as of anoikis resistance. We identified the interactions of SqE with glycogen synthase kinase 3β (GSK3β) and/or p53: SqE knockdown induced the dissociation of GSK3β and p53, thereby resulting in the downregulation of E-cadherin via the inhibition of GSK3 activity and of -catenin degradation, as well as the p53 degradation by the hdm2 upregulation. Overall, the present study demonstrates SqE is a key negative regulator of cancer cell survival and of CRC metastasis.

Citation Format: Soo Young Jun, Hyun-Soo Cho, Jeong-Ju Lee, Ji-Yong Yoon, Jun-Ho Ahn, Ju-Sik Min, Sujin Jeon, Jae-Hye Lee, Min Hyuk Choi, Nam-Soon Kim. A novel tumor-suppressor role for squalene epoxidase in human colorectal 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 4130.
Abstract 4134: Immune infiltration and PD-L1 expression in the tumor microenvironment are prognostic in osteosarcoma
Authors: Koirala Pratistha , Roth Michael , Gill Jonathan , Chinai Jordan , Piperdi Sajida , Geller David , Hoang Bang , Poon Vincent , Zang Xingxing , Gorlick Richard
Published: July 2016

Purpose: Over the past four decades, osteosarcoma (OS) survival rates have remained stagnant. There is a need to identify novel therapies to target OS. In this study we examined the expression of Programed Death Ligand 1 (PD-L1) and defined the tumor microenvironment in OS in order to assess the feasibility of utilizing immune checkpoint inhibitors as a treatment modality.

Experimental Design: PD-L1 expression in OS was examined in two patient cohorts using immunohistochemistry (IHC) (n = 48, n = 59) and expression was validated using quantitative real time PCR (qRT-PCR) (n = 21) and western blotting (n = 9). IHC was used to determine presence of tumor infiltrating lymphocytes and antigen presenting cells (APCs) in the tumor. Expression of PD-L1 was correlated with the presence of immune cells and with event free survival in OS.

Results: PD-L1 is expressed in up to 25% of primary OS tumors. Of the PD-L1 positive tumors the majority were also PD-1 positive (92% vs 8%, p = 0.002). In addition, the presence of immune cells in the tumor mass was significantly associated with PD-L1 expression. Although all immune cell types examined were present in OS, only infiltration by APCs, specifically CD1a positive dendritic cells (48.6% vs. 51.4%, p = 0.0010) and CD68 positive macrophages (70.3% vs. 29.7%, p = 0.0316), was associated with worse outcomes. PD-L1 expression was significantly associated with worsened survival (21.6% pos. vs. 78.4% neg., p = 0.0146).

Conclusions: For the first time we have identified PD-L1 expression or presence of CD1a or CD68 positive antigen presenting cells as prognostic markers for worsened outcome in OS. Furthermore, we show that IHC is a valid detection method for PD-L1 in OS. With up to 25% of OS patients expressing PD-L1, this study provides rational for targeting the PD-L1:PD-1 axis for immunotherapy in OS.

Citation Format: Pratistha Koirala, Michael Roth, Jonathan Gill, Jordan Chinai, Sajida Piperdi, David Geller, Bang Hoang, Vincent Poon, Xingxing Zang, Richard Gorlick. Immune infiltration and PD-L1 expression in the tumor microenvironment are prognostic in osteosarcoma. [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 4134.
Abstract 414: Biochemical and cellular monitoring of the activity of the ecto-5’-nucleotidase (CD73), a key cancer modulator using HTS-formatted bioluminescent technology
Authors: Goueli Said A. , hsiao. Kevin
Published: July 2016

The ecto-5’-nucleotidase (CD73) is glycosylphosphatidylinositol (GPI) anchored cell surface protein that is involved in switching on adenosinergic signaling. Its enzymatic and nonenzymatic activities (via its interaction with extracellular matrix components) are involved in cancer associated processes and not completely independent of each other. It catalyzes the hydrolysis of AMP into adenosine and phosphate, where adenosine plays important role in tumor immune escape. It is overexpressed in many types of cancer cell lines and patient's biopsies including breast, colon, ovarian, gastric, ovarian, etc. In addition to being important as clinical and prognostic marker in cancer patients, overexpression of CD73 is associated with resistance to antitumor agents, and inhibition of CD73 activity or knocking it down by SiRNA reversed chemoresistant phenotype of glioblastoma multiforme cells. Because of the significant role of CD73, it is of importance to develop an assay that monitor the activity of CD73 in order to develop modulators of its activity. Towards this goal, we have developed a bioluminescent assay to monitor the activity of the enzyme in biochemically pure as well cellular anchored enzyme forms. The assay is homogenous and formatted for HTS screening research, very sensitive, and robust as indicated by the high Z’. We also show that the use of inhibitors such as adenosine 5’-(α-β-methylene) diphosphate (APCP) generates data with biochemically pure enzyme similar to the cellular bound form. We also demonstrate that cells that are enriched in CD73 can be easily identified from those that have low CD73. Thus, the current assay is robust, sensitive, and easy to use making it an ideal assay for HTS screening for new modulators of CD73 and generation of potential novel cancer therapeutics

Citation Format: Said A. Goueli, Kevin hsiao. Biochemical and cellular monitoring of the activity of the ecto-5’-nucleotidase (CD73), a key cancer modulator using HTS-formatted bioluminescent technology. [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 414.
Abstract 4142: Proteomics analysis of breast cancer cell-specific proteins that are transferred to immune cells via trogocytosis
Authors: Suzuki Eiji , Inoue Masashi , Ito Shinji , Satoh Junko , Kawaguchi Kosuke , Yamaguchi Ayane , Tsuda Moe , Toi Masakazu
Published: July 2016

We previously reported that HER2 is transferred from HER2 overexpressing breast cancer cells to CD14+ monocytes via trastuzumab dependent trogocytosis (Suzuki et al. BMC cancer 2015) and EGFR of MDA-MB-231 (triple negative human breast cancer cell line) is also transferred to immune cells via trogocytosis and those immune cells express EGFR on the cell surface without therapeutic antibody administration (AACR 2015 #2349). There are several reports that suggest that trogocytosis might act to stimulate immunological tolerance or immune effector cell activation, however its immunological roles still remain unclear. Therefore, in order to clarify the role of trogocytosis, we study the profile of proteins that potentially are transferred from cancer cell to immune cell via trogocytosis.

Human triple negative breast cancer cell line, MDA-MB-231 that was cultured with the medium containing arginine labeled with 13C instead of normal 12C (SILAC method, ThermoFisher Scientific) was co-cultured with human peripheral blood mononuclear cell (PBMC) that was cultured with normal medium. The cell mixture was labeled with CD45+ magnetic beads and CD45+ cell (PBMC) and CD45- cell (MDA-MB-231) were separated by MACS cell separation kit (Miltenyi Biotec). Proteins of PBMC that were passed the trogocytosis were resolved on SDS-PAGE and LC-MS/MS was carried out to identify the cancer specific trogocytosed proteins in PBMC by SILAC method.

We found that proteins with higher H/L ratio were included in many of cytoskeletal proteins and interestingly mitochondrial metabolism related proteins of cancer cells were also detected in PBMC. Those up-regulated proteins in PBMC were reduced in MDA-MB-231. The findings suggested that the role of trogocytosis of PBMC on breast cancer cell might be involved in metabolisms of both cancer cell and immune cell. Thus, we are exploring the effect of trogocytosis on cancer cell metabolisms especially on mitochondrial related metabolism that could be reduced by immune cell trogocytosis and also on metabolism of immune cells themselves that capture the cancer cell proteins via trogocytosis.

Citation Format: Eiji Suzuki, Masashi Inoue, Shinji Ito, Junko Satoh, Kosuke Kawaguchi, Ayane Yamaguchi, Moe Tsuda, Masakazu Toi. Proteomics analysis of breast cancer cell-specific proteins that are transferred to immune cells via trogocytosis. [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 4142.
Abstract 4156: Functional analysis of tumor-associated macrophage utilizing virus-guided fluorescent imaging of pancreatic cancer cells in the peritoneal cavity
Authors: Kuwada Kazuya , Kagawa Shunsuke , Watanabe Megumi , Sakamoto Shuichi , Kikuchi Satoru , Kuroda Shinji , Yoshida Ryuichi , Tazawa Hiroshi , Kagawa Tetuya , Fujiwara Toshiyoshi
Published: July 2016

Objectives:

Postoperative recurrence occurs in approximately 80% of pancreatic cancer, and the peritoneum is the second most frequent metastatic site next to the liver. Recent evidence suggests that cancer microenvironment plays key roles in metastasis. To investigate mechanism of intraperitoneal metastasis in pancreatic cancer, we tried to detect cancer cell in the peritoneal wash from pancreatic cancer patients and analyze intraperitoneal cancer microenvironment. A genetically engineered adenovirus, TelomeScan, which replicates and expresses GFP only in telomerase-active cancer cells, was employed to detect cancer cells, and it enabled us to distinguish cancer cell from co-existence cells in the abdominal cavity. We explored the role of tumor-associated macrophages (TAMs) by using this virus-guided fluorescent imaging system.

Methods:

Peritoneal wash was obtained from 20 pancreatic cancer patients during operation. The cells in the wash were infected with TelomeScan for 24 hours. Samples from cases with TelomeScan-expressing GFP-positive cells were further subjected to immunofluorescence assay for analysis of microenvironment. The antibodies against CD45, CD14, and CD204 were used in immunostaining as markers of leukocytes, monocytes and TAMs, respectively. To investigate the effect of TAMs on pancreatic cancer, Panc1 and BxPC-3 cell lines were co-cultured with PMA (phorbol 12-myristate 13-acetate)-treated monocytes and analyzed their changes.

Results:

The three out of 20 cases were positive in cytology, and GFP positive cells were detected after TelomeScan infection in those cases. In the wash, pancreatic cancer cells existed together with many leukocytes including macrophages. Further analysis demonstrated that those macrophages were TAMs. After Panc1 or BxPC-3 cells were co-cultured with TAMs, E-cadherin was decreased in Panc1, α-SMA and Vimentin was increased in BxPC-3. These results suggested that Epithelial to Mesenchymal Transition (EMT) was induced in pancreatic cancer cells by TAMs.

Conclusion:

Pancreatic cancer cells and TAMs were detected in the peritoneal wash using TelomeScan and immunostaining. The results suggested that EMT induced by TAMs may promote intraperitoneal metastasis of pancreatic cancer.

Citation Format: Kazuya Kuwada, Shunsuke Kagawa, Megumi Watanabe, Shuichi Sakamoto, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroshi Tazawa, Tetuya Kagawa, Toshiyoshi Fujiwara. Functional analysis of tumor-associated macrophage utilizing virus-guided fluorescent imaging of pancreatic cancer cells in the peritoneal cavity. [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 4156.
Abstract 4163: Molecular and physiological effects of splicing factor mutant U2AF1 in human lung cell lines and in mice
Authors: Fei Dennis , Motowski Hayley , Prasad Sameer , Yu Jovian , Bradley Robert , Varmus Harold
Published: July 2016

The splicing factor gene, U2AF1, is commonly mutated in myeloid neoplasms and occasionally in solid tumors such as lung adenocarcinomas (LUADs), yet the molecular and physiological consequences of U2AF1 mutations are not well understood. Through genome editing of the endogenous locus, we created a common U2AF1 mutant allele (U2AF1-S34F) in an immortalized human bronchial epithelial cell line, and we inactivated the U2AF1-S34F allele in two LUAD cell lines that naturally harbor this mutation. Cells expressing U2AF1-S34F deployed altered splicing of many transcripts, and they displayed a S34F-characteristic sequence pattern at the proximal 3’ splice site, as previously reported. Remarkably, S34F-associated alternative splicing patterns remained unaffected by changes in the amount of mutant U2AF1 protein, as long as the ratio of mutant and wild-type U2AF1 remained constant, as evidenced by RNA interference-mediated knockdown of the expression of both mutant and wild-type alleles. On the other hand, perturbing the ratio of mutant and wild-type U2AF1, either through overexpression or allelic-specific inactivation of wild-type U2AF1, greatly affected S34F-associated splicing. These results suggest that the ratio of mutant and wild-type U2AF1, rather than the absolute amount of either protein, is critical in controlling splicing outcome. Expression of U2AF1-S34F does not cause cell transformation and LUAD cells with endogenous U2AF1-S34F expression are not dependent on the U2AF1-S34F allele. Instead, wild-type U2AF1 is absolutely required for cell survival in the presence of the mutant allele. To study the effect of U2AF1-S34F in vivo, we created transgenic mice carrying a conditional mutant allele, MG-S34F, at the endogenous U2af1 locus. By crossing the MG-S34F mice with mice carrying a Mx1-Cre transgene and treating the progeny with poly IC, we activated MG-S34F by Cre-mediated recombination to express U2af1-S34F in bone marrow cells. This resulted in cytopenia in multiple blood cell lineages. Similar results were observed in recipient mice following non-competitive transplant. Bone marrow cells expressing U2af1-S34F were competitively disadvantaged in repopulating the hematopoietic system in irradiated mice, suggesting defective hematopoietic stem cell function, and we are currently assessing the long-term impact of U2af1-S34F expression on the incidence of myeloid neoplasms.

Citation Format: Dennis Fei, Hayley Motowski, Sameer Prasad, Jovian Yu, Robert Bradley, Harold Varmus. Molecular and physiological effects of splicing factor mutant U2AF1 in human lung cell lines and in 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 4163.
Abstract 4166: Role of calreticulin in the spontaneous development of lung adenocarcinoma: Evidence from a novel transgenic mouse model
Authors: Mesaeli Nasrin , Massaeli Hamid , Viswanathan Divya , Pillai Dhanya , Thomas Mercy Anna
Published: July 2016

Lung cancer is the most common cause of cancer related deaths in both men and women worldiwide. The overall 5 year survival rate of lung cancer ranges between 5-15% (depending on the subtype). Based on histopathological criteria, lung cancer is subdivided into two main subtypes: small cell lung cancer (15%) and non-small cell lung cancer (NSCLC 85%). Adenocarcinoma a subtype of NSCLC is the most common type in both sexes, and it has a poor response to chemotherapy. We recently developed a transgenic mouse model of lung adenocarcinoma by overexpressing calreticulin (CRT) under the control of Tie2-promotor. The main phenotype of these mice is high incidence of metastatic lung adenocarcinoma similar to human patients. Immunohistological analysis showed these tumors to be SP-C+ve and CC10-ve phenotype. At the initial stages most of cells in the hyperplegic area and adenoma stained positive for exogenous CRT and HSCs markers, and as the tumor differentiated to carcinoma there were a significant loss of exogenous CRT and HSCs markers and increased expression of SP-C.

The aim of our current project was therefore to identify the mechanism of development of metastatic lung adenocarcinoma in this mouse model. To address this objective we carried out microarray and quantitative proteomic analysis of tissue and cell lines isolated from lungs of this transgenic mouse model.

Microarray analysis showed activation of cluster of genes involved in cellular development, growth and proliferation. Some of these pathways include genes invlved in VEGF signal transduction pathways and Epherin receptor signaling, at the same time there were decreased expression of cell-cell interaction and communication (needed for metastasis). At the protein level we observed a significant increase in proteins involved in tumor progression, invasion and metastasis. In addition we observed a significant change in the expression of genes invloved in lipid metabolism (both at protein and RNA level) in the lung of these mice. The change in the expression of lipid metabolizing genes suggest the metabolic shift needed during tumor progression.

In conclusion, our data suggests that CRT as an endoplasmic reticulum chaperone and a regulator of intracellular calcium homeostasis affects multiple genes invivo resulting in the development of tumor.

This research was funded by Qatar National Research Fund (NPRP4-043-3-016).

Citation Format: Nasrin Mesaeli, Hamid Massaeli, Divya Viswanathan, Dhanya Pillai, Mercy Anna Thomas. Role of calreticulin in the spontaneous development of lung adenocarcinoma: Evidence from a novel transgenic mouse model. [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 4166.
Abstract 4168: Mig-6 ablation cooperates with oncogenic Kras in promoting mouse lung tumorigenesis
Authors: Liu Jian , Cho Sung-Nam , Jin Nili , Moghaddam Seyed Javad , Gilbert Jennifer , Wistuba Ignacio , DeMayo Francesco J.
Published: July 2016

Lung tumorigenesis is a stochastic multistep process, during which different gene mutations accumulate, and Kras is an oncogene with the most frequent mutation in the Caucasian lung adenocarcinoma. Although Mig-6 (mitogen-inducible gene 6) has been shown to be a tumor suppressor gene in lung, skin and uterus, the interplay between Mig-6 and Kras in lung tumorigenesis remains elusive. Here, we found Mig-6 expression was down-regulated in oncogenic KrasG12D-induced lung tumors and Mig-6d/dKrasG12D mice showed an earlier onset of pulmonary tumor development and a significantly reduced life span as compared to KrasG12D mice though no obvious lung phenotype was observed in Mig-6d/d mice. Meanwhile, ablation of Mig-6 can change the lung epithelial cell fate in KrasG12D mice. Furthermore, lung tumor tissues of Mig-6d/dKrasG12D mice showed decreased cellular apoptosis, elevated EGF/Akt signaling and increased severity of inflammation as compared to those of KrasG12D mice. Thus, ablation of Mig-6 cooperated with oncogenic Kras in promoting lung tumorigenesis and this novel murine model of lung cancer can be applied for future study.

Citation Format: Jian Liu, Sung-Nam Cho, Nili Jin, Seyed Javad Moghaddam, Jennifer Gilbert, Ignacio Wistuba, Francesco J. DeMayo. Mig-6 ablation cooperates with oncogenic Kras in promoting mouse lung tumorigenesis. [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 4168.
Abstract 4172: A role for RAD52 as a lung cancer susceptibility gene in the 12p13.33 locus
Authors: Lieberman Rachel , Xiong Donghai , You Ming
Published: July 2016

Although 80-90% of lung cancer patients smoke, only about 10% of heavy smokers develop lung cancer. This suggests that although tobacco smoke undoubtedly increases one's risk for lung cancer, genetic risk factors most likely predispose certain individuals. Recent genome-wide association studies identified variation in the recombination repair gene, RAD52, associated with increased lung cancer risk, and particularly with squamous cell lung carcinoma (SCC). SCC development is strongly associated with smoking and individuals who smoke have increased DNA repair, presumably to address ongoing DNA damage from exposure to tobacco smoke. Hence, it fits that the genomic region of 12p13.33 containing RAD52 shows both copy number amplification and an increase in gene expression in SCC tumors compared to normal. Functionally, we have previously demonstrated that overexpression of Rad52 in mouse bronchial epithelial and lung tumor cells leads to an increase in growth rate and that depletion leads to decreased cell growth, senescence, and an accumulation of cells in G2/M.

Our current study focuses on genetic instability within lung tumor progression and how the DNA repair gene RAD52 allows primary tumor cells to become genetically stable enough to be able to invade and avoid apoptosis. C57BL6 wildtype or Rad52−/− mice at 8 weeks of age were randomized into two groups and treated topically with 0.04 M NTCU in 100-microliter drop, twice a week for 40 weeks. Unlike mouse lung adenocarcinomas, mouse SCC does not form visible solid nodules on the surface of the lung. Lungs were fixed and stained with H&E and examined histologically under a light microscope to establish tumor multiplicity and the types of lesions (invasive SCC, SCC in situ, or bronchial hyperplasia/metaplasia). Lung tissue was also stained for TUNEL analysis as well as markers of SCC and proliferation. Our novel findings demonstrate that depletion of Rad52 in vitro generates increased DNA damage and sensitivity to cell death, while knockout of the Rad52 gene in an in vivo mouse model of carcinogenesis decreases murine lung hyperplasia, in situ carcinoma and lung SCC. In addition, knockout appears to increase apoptosis. Thus, our functional studies continue to support genetic findings by revealing that loss of RAD52 can enhance cell death and DNA damage in lung tumor cells. This complementary evidence further strengthens the notion of RAD52 as a potential oncogene and implicates a major role for the process of recombinational repair in determining risk for SCC.

Citation Format: Rachel Lieberman, Donghai Xiong, Ming You. A role for RAD52 as a lung cancer susceptibility gene in the 12p13.33 locus. [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 4172.
Abstract 4176: Leptin mediates the anti-breast cancer effects of environmental enrichment
Authors: Foglesong Grant , Haung Wei , Cao Lei
Published: July 2016

Obesity affects 35% of U.S. adults and is a leading risk factor for breast cancer in postmenopausal women, but worsens prognosis regardless of menopausal status. Importantly, progression to obesity can largely be slowed or reversed with aggressive lifestyle changes, thus also having the potential to mitigate cancer onset. A mouse model of environmental enrichment (EE) to improve motosensory, cognitive, and social stimulation by increasing physical engagement and social interaction triggers vast improvements in overall health. These include reducing adiposity, promoting the white to brown fat transition, mitigating diet-induced obesity (DIO), and decreasing progression of multiple cancer types including colon cancer and melanoma. We have elucidated the primary mechanism of the EE-induced phenotype to be the activation of the hypothalamic-sympathoneural-adipocyte (HSA) axis, a specific neuroendocrine route in which the brain communicates with adipose tissue. These benefits may also be partially attributable to the sharp drop in serum leptin levels following EE which has been implicated in diminishing tumorigenesis, invasiveness, and metastasis. Here we investigated the effects of EE on breast tumorigenesis in all body mass states and menopause-associated hormone conditions. Our preliminary studies showed that EE delayed the cancer onset in the MMTV-PyMT spontaneous mouse model of breast cancer. In addition the role of leptin signaling in the EE-induced effects on breast tumorigenesis was investigated by utilizing obese models with varied leptin signaling including leptin receptor-defective db/db mice that express extremely high levels of leptin, leptin-defective ob/ob mice that do not express leptin but are leptin-sensitive, and a diet-induced obesity (DIO) model with elevated leptin but intact leptin signaling. These obese mice were housed in EE or control housing until significantly attenuated weight gain was observed followed by implantation of primary PyMT-derived mammary tumor cells. EE was highly effective at reducing adiposity in each of these obesity models, regardless of leptin signaling. However, the effects of EE on mammary tumor progression were dependent on leptin signaling. EE decreased leptin level and inhibited mammary tumor growth in DIO mice. In contrast EE failed to attenuate tumor progression in ob/ob mice in the absence of leptin, suggesting that leptin is a key mediator of the EE anti-cancer effects on breast cancer. The elucidation of mechanisms of EE and the HSA axis-induced improvement in overall health will provide effective prevention as well as therapeutic strategies for metabolic syndromes and associated cancer types.

Citation Format: Grant Foglesong, Wei Haung, Lei Cao. Leptin mediates the anti-breast cancer effects of environmental enrichment. [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 4176.
Abstract 4180: Rewiring the cytokine network in melanoma
Authors: Nagaraju Raghavendar , Dee Christopher , Young Helen , Barriuso Jorge , Secombes Christopher , Hurlstone Adam
Published: July 2016

Malignant melanoma is one of the most aggressive types of malignancies in humans resulting in 50,000 deaths worldwide annually. Fortuitously, malignant melanoma is highly immunogenic and it is now well established that the host immune system can detect and kill melanoma. T helper type-1 lymphocytes (TH1) play a pivotal role in inducing and maintaining anti-tumour immune responses. In melanoma patients, it has been observed that higher number of TH1 cells in the tumour micro-environment lead to better prognosis. In this study, using an autochthonous zebrafish melanoma model, when we forced the expression of interferon gamma (IFNg) locally in the tumour microenvironment, thereby potentially enhancing TH1 differentiation, we observed an inflammatory response against melanoma that eventually lead to complete tumour regression.

Using transposon mediated BAC transgenesis, we have generated a Tg(cd4:mcherry) reporter line labelling CD4+ cells in zebrafish. By driving Human NRASQ61L in zebrafish melanocytes we have modelled melanoma in zebrafish and later when these fish developed tumours we forced the expression of IFNg in melanoma cells using a tamoxifen inducible LSL/CreERT2 system driven by mitfa minimal promoter. Experiments were performed using animals with and without CreERT2. Following tamoxifen administration, we observed a loss of pigmentation in all the IFNg induced tumours (n = 12) by 2 weeks post induction which gradually increased over the study period. Whereas no loss of pigmentation was observed in control tumours (n = 12) throughout the study period. At 9 weeks post induction, the study was terminated and tumours were analysed using histology. In tumours were IFNg was induced, we observed that the tumour tissue was now replaced with fibrotic tissue accompanied by a marked lymphocyte infiltration and patches of necrosis. However, new tumour nodules were also observed to develop in the vicinity of regressing nodules which are unpigmented and potentially hypo-immunogenic. We observed no signs of tumour regression in control tumours.

Although preliminary, our data is very exciting and promising as it will open new avenues for developing combinatorial therapies in melanoma. Using a high throughput in-vivo model system such as zebrafish, it is an exciting proposition to test the impact of combining anti-PD1/PDL1 inhibitors with various cytokines/chemokines in inducing effective and sustained tumour regression in melanoma.

Citation Format: Raghavendar Nagaraju, Christopher Dee, Helen Young, Jorge Barriuso, Christopher Secombes, Adam Hurlstone. Rewiring the cytokine network in 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 4180.
Abstract 4188: RNAi and CRISPR/Cas9 based in vivo models for drug discovery
Authors: Premsrirut Prem K. , Martin Gregory , Dow Lukas , Kim Sang Yong , Zuber Johannes , Lowe Scott , Hannon Greg
Published: July 2016

With the advent of CRISPR-Cas9 technology, the speed and precision in which genetically engineered mouse models can be created is unprecedented. We now have at our disposal a genetic toolbox that will enable the rapid generation of sophisticated mouse models of cancer. Recently, an inducible CRISPR-Cas9 (iCRISPR) system was described that enables doxycycline-regulated Cas9 induction of widespread gene mutagenesis in multiple tissues. Previously, we also demonstrated how inducible RNA interference (RNAi) can be exploited experimentally to effectively and reversibly silence nearly any gene target not only in vitro but also in live mice. Here, we take advantage of these powerful technologies and combine both tet-inducible CRISPR-Cas9 and inducible RNAi-mediated gene silencing to develop animal models in which both de novo tumorigenesis can be induced by Cas9-mediated genome editing and therapeutic strategies assessed downstream via RNA interference-mediated gene silencing. By using this combination of CRISPR/Cas9 and RNAi technologies, we are able to not only model disease pathogenesis, but also mimic drug therapy in the same mice, giving us exceptional capabilities to perform preclinical studies in vivo. Using our robust flexible system, we have created a cost-effective and scalable platform for the production of complex genetically engineered mouse models of cancer with RNAi silencing of nearly any gene - mice with enormous predictive power that will shape our development of better tolerated therapies.

Citation Format: Prem K. Premsrirut, Gregory Martin, Lukas Dow, Sang Yong Kim, Johannes Zuber, Scott Lowe, Greg Hannon. RNAi and CRISPR/Cas9 based in vivo models for drug discovery. [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 4188.
Abstract 4193: Reporter systems to measure dynamics and specificity of targeted cancer therapy
Authors: Marquez Rebecca T. , Xu Liang ,   Experimental and Preclinical Imaging Core
Published: July 2016

The Precision Medicine Initiative has driven the need for targeted therapy development to increase cancer patient survival. Due to this initiative, developing enhanced methods for measuring the accuracy and specificity of lead compound delivery is critical for moving these compounds through preclinical studies at a more efficient rate. The Experimental and Preclinical Imaging Core at the University of Kansas (epicore.ku.edu) is developing reporter constructs to assist in validating lead compound inhibition of oncogenic targets. Our lentiviral reporter constructs are used to establish stable cell lines that can be used either in vitro or in vivo to ensure optimal target inhibition. Our constructs include promoter reporters as well as 3’ untranslated-region (UTR) reporters to measure either changes in mRNA stabilization or translation by RNA-binding proteins or microRNA target validation. Our goal is to provide reporter constructs that will enable industry and academic institutions to ensure optimal inhibition of signaling pathways targeted by their lead compounds or microRNA-based therapies. Currently, we are constructing stable cancer cell lines that express near-infrared proteins, such as iRFP670, for measuring primary tumor and metastatic growth in vivo. In comparison to luciferase-expressing cell lines, we have found that cells expressing iRFP670 provided a more robust signal and were easier to image without the need for injecting a substrate. New promoter and 3’UTR reporters are being developed using iRFP670 and will be tested in vivo.

Citation Format: Rebecca T. Marquez, Liang Xu, Experimental and Preclinical Imaging Core. Reporter systems to measure dynamics and specificity of targeted cancer 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 4193.