bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5002: Anti-tumor activity of a novel anti-human PD-1 antibody BGB-A317 in mouse models
Authors: Song Xiaomin , Tang Zhiyu , Zhang Tong , Guo Mingming , Gong Wenfeng , Liu Yong , Zhang Ningning , Zhang Yilu , Zhang Yanjuan , Ma Jie , Song Jing , Li Kang , Wang Lai
Published: July 2016

Background: Programmed death receptor-1 (PD-1) is a co-inhibitory immune checkpoint molecule. The engagement of PD-1 by its ligands negatively regulates T cell function and induces immune tolerance in both normal and pathological conditions, especially in cancer. BGB-A317 is a novel humanized IgG4 anti-PD-1 antibody under clinical development. The anti-tumor activity of BGB-A317 was evaluated in mouse models and reported here.

Material and methods: NOD/SCID mice transplanted with human peripheral blood monocyte (PBL-SCID models) were employed to examine the anti-tumor activity and pharmacokinetics of BGB-A317. In these models, human PBMCs (4-5×106) from healthy donors were co-injected with human tumor cells (A431, 2.5×106) or primary tumor tissue fragments derived from cancer patients (BCCO-028 and BCLU-054, 3×3×3 mm3) subcutaneously in the right flank of the mice. BCCO-028 is human colon cancer and BCLU-054 is human non-small cell lung cancer (NSCLC). The mice were then treated with BGB-A317 or PBS intra-peritoneally once a week from the day of tumor implantation. The tumor infiltrated lymphocytes and PD-L1 expression were examined by immunohistochemistry staining and flow cytometry.

Results: In A431 model, 3 doses of BGB-A317, 1, 3, 10mg/kg, were examined. All three doses of BGB-A317 induced significant tumor growth inhibition (TGI) on day 29 (78%, 89%, and 89%, respectively) (p < 0.001 vs. vehicle treatment). Both AUC0-168h and Cmax of BGB-A317 after single dose (day 1, 1st dose) or at steady state (day 29, 5th dose) increased proportionally from 1 to 10 mg/kg. There was about 2-fold accumulation in drug levels (AUC0-168h and Cmax) at the steady state compared to those after single dose. Increased lymphocyte infiltration and PD-L1 expression were observed in BGB-A317 treated groups. In BCCO-028 model, BGB-A317 at 10 mg/kg QW induced significant tumor growth inhibition, with 83% TGI (p < 0.05 vs. vehicle treatment) on day 36. In BCLU-054 model, BGB-A317 at 10 mg/kg QW induced significant tumor growth inhibition, with 45% of TGI (p < 0.05 vs. vehicle treatment) on day 23. BGB-A317 treatment had no significant impact on animal body weight throughout all of the studies.

Conclusions: BGB-A317 demonstrated significant anti-tumor activity in 3 human cancer xenograft PBL-SCID mouse models, including A431 human epidermoid carcinoma, BCCO-028 colon cancer, and BCLU-054 NSCLC models.

Citation Format: Xiaomin Song, Zhiyu Tang, Tong Zhang, Mingming Guo, Wenfeng Gong, Yong Liu, Ningning Zhang, Yilu Zhang, Yanjuan Zhang, Jie Ma, Jing Song, Kang Li, Lai Wang. Anti-tumor activity of a novel anti-human PD-1 antibody BGB-A317 in mouse models. [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 5002.
Abstract 5008: Elucidating HER2 molecular heterogeneity of circulating tumor cells among breast cancer patients
Authors: Leong Man Chun , Lim Tse Hui , Tan Chye Ling , Chua Yong Wei , Lim Elaine Hsuen , Loh Kiley Wei Jen , Lee Guek Eng , Dent Rebecca , Ng Raymond Chee Hui , Wu Andrew , Lim Wan-Teck , Lim Alvin Soon-Tiong , Yap Yoon-Sim
Published: July 2016

Background: HER2-positive tumors are often associated with poor prognosis, chemo-resistances and some patients eventually develop refractory disease during HER2 targeted therapy. While different mechanisms of trastuzumab resistance are being identified in recent years, contribution of confounding factors such as inherent genomic heterogeneity, equivocal HER2 amplifications and presence of chromosome 17 polysomy has been less understood thus far. In this pilot study, we aim to examine HER2 heterogeneity in CTCs obtained from breast cancer patients in the Asian population setting.

Methods: CTCs were enriched from blood samples using a label-free spiral microfluidics-based ClearCell® FX system. A total of 26 samples were collected from patients diagnosed with HER2 positive (17/26) and HER2 negative (9/26) breast cancer. The enriched CTCs were analyzed using conventional diagnostic modalities (fluorescence in-situ hybridisation (FISH) and immunocytochemistry) to examine HER2 status. Concordance rate between CTCs and matched primary tumor was evaluated.

Results: HER2-positive CTCs were successfully identified in 14 out of 17 HER2+ patients (82.4%); HER2 gene amplification and chromosome 17 polysomy were observed in 10(58.8%) and 13(76.5%) patients respectively. HER2+ CTC counts ranged from 2 to 30 cells from 7.5ml blood (median: 4 HER2+ CTCs/7.5ml). HER2 amplification was not observed in any of the 9 patients with HER2-negative tumors, though 5 out of 9 patients (55.6%) were identified with CTCs harbouring gain in chromosome 17 (median: 2 HER2+ CTCs/7.5ml). A “false positive” cut-off of more than 2 cells/7.5ml blood were established using receiver operating characteristic (ROC) curve analysis and a concordance rate of approximately 70% between paired tumor tissue and CTC among the 26 patients. Immunofluorescence labelling of CTCs with cytokeratin, CD45 and HER2 antibodies further revealed heterogeneity in HER2 expression on CTCs.

Conclusion: CTCs capture the heterogeneity of breast cancer, and could potentially overcome limitations of tissue biopsy which are site specific. HER2- patients, as confirmed by tissue biopsy, with HER2+ CTCs pose interesting questions while determining treatment regime.

Citation Format: Man Chun Leong, Tse Hui Lim, Chye Ling Tan, Yong Wei Chua, Elaine Hsuen Lim, Kiley Wei Jen Loh, Guek Eng Lee, Rebecca Dent, Raymond Chee Hui Ng, Andrew Wu, Wan-Teck Lim, Alvin Soon-Tiong Lim, Yoon-Sim Yap. Elucidating HER2 molecular heterogeneity of circulating tumor cells among breast cancer 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 5008.
Abstract 5030: Tumor necrosis serves as a prognostic predictor in operable and stage I lung adenocarcinoma
Authors: Lin Pei-Ying , Hsiao Tzu-Hung , Yang Pan-Chyr
Published: July 2016

Background

A relatively high proportion of stage I lung adenocarcinoma patients suffer from disease relapse after surgical resection. This highlights an unmet need in clinical risk assessment for this patient group. Several previous studies suggested adopting the feature tumor necrosis as a risk stratification parameter, but consensus has not been reached. Here we evaluated whether gene signatures that represented three biological processes, cell proliferation, tissue hypoxia and nutrient supplement, underlying their pathological sum tumor necrosis, supported the assumption that necrosis is a prognostic predictor.

Methods and Results

The three selected gene signatures, cell-cycle, hypoxia and mTOR, were revealed to have higher signature activities in necrosis-positive lung adenocarcinoma tissues than in necrosis-negative ones. This phenomenon was observed in a Japanese dataset and was verified in The Cancer Genome Atlas lung adenocarcinoma dataset. In these two datasets, both the feature tumor necrosis and the signatures were shown to stratify operable and/or stage I lung adenocarcinoma patients into different risk groups. We further validated this observation in a 130 surgically resected lung adenocarcinoma patient cohort. The results showed that tumor necrosis predicted shorter overall and relapse-free survival in patients with operable (P<0.001 and P = 0.007, respectively) and stage I (P = 0.003 and P = 0.03, respectively) lung adenocarcinomas.

Conclusions

Our study showed that tumor necrosis served as a prognostic predictor in operable and stage I lung adenocarcinomas. The findings supported the results from previous pure pathological studies.

Citation Format: Pei-Ying Lin, Tzu-Hung Hsiao, Pan-Chyr Yang. Tumor necrosis serves as a prognostic predictor in operable and stage I lung adenocarcinoma. [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 5030.
Abstract 5031: Melanoma progression involves a profound nuclear to cytoplasmic shift of the transcription factor SUM-6 (specifically upregulated in melanoma gene six)
Authors: Graziano Laura J. , Zhang Xue , Cheng Yabin , Wang Gang , Su Mingwan , Martinka Magdalena , Zhou Youwen
Published: July 2016

INTRODUCTION: SUM-6 is a human homeobox gene that encodes for a transcription factor, which plays a key role in normal embryogenesis. Overexpression of both the gene and nuclear protein has been shown to occur in a variety of cancers such as breast, colorectal, and pancreatic, and has been demonstrated to promote tumorigenesis. The purpose of the current study was to evaluate the expression of the SUM-6 protein, its clinical relevance, and biological function in melanoma.

EXPERIMENTAL PROCEDURES: Immunohistochemistry using a specific SUM-6 antibody was performed on a tissue microarray consisting of 438 patient biopsies, which included benign and malignant melanocytic tumors. Double-blinded scoring of distinct nuclear and cytoplasmic SUM-6 staining was performed. SPSS 16.0 statistical package and Microsoft Excel were used to carry out the analyses of data. Analyses included the χ2 test and univariate analysis by the log-rank test in conjunction with the Kaplan-Meier survival curve for visualization.

RESULTS: Nuclear staining of SUM-6 was detected in 100% of normal nevi (n = 19), 81% of dysplastic nevi (n = 32), 87% of melanoma in situ (n = 23), 79% of primary melanoma (n = 219), and 53% of metastatic melanoma (n = 145), whereas cytoplasmic staining was detected in 63%, 94%, 56%, 94%, and 97%, respectively. Univariate analysis revealed that five year disease-specific survival decreased significantly when SUM-6 was excluded from the nucleus and when SUM-6 levels were elevated in the cytoplasm (p = 0.001 and 0.019, respectively).

CONCLUSIONS: We concluded that melanoma progression and metastasis is associated with a profound shift of transcription factor SUM-6 from the nucleus to the cytoplasm. It is interesting to note that in most other cancers, the SUM-6 protein was instead overexpressed in the nucleus. The patients in whom SUM-6 was excluded from the nucleus demonstrated a significantly worse prognosis than the patients who retained nuclear expression. These findings suggest that either the absence of nuclear expression or elevated cytoplasmic presence (or both) of SUM-6 may play a role in the progression of malignant melanoma.

Citation Format: Laura J. Graziano, Xue Zhang, Yabin Cheng, Gang Wang, Mingwan Su, Magdalena Martinka, Youwen Zhou. Melanoma progression involves a profound nuclear to cytoplasmic shift of the transcription factor SUM-6 (specifically upregulated in melanoma gene six). [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 5031.
Abstract 5042: A CRISPR/Cas9 knockout screen identify novel drug resistance genes in AML
Authors: Huang Jian
Published: July 2016

Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. Despite remarkable progress made in the therapy of AML, the mainstays of treatment have not significantly changed over the last 20 years. Technological advances have greatly accelerated genomic discovery and precision medicine holds promise to better tailor medications to AML with specific mutations. Importantly, one major obstacle to greater success with target therapy of leukemia is drug resistance. But the mechanisms underlying drug resistance in AML are poorly understood.

The activating mutations of FLT3 are now recognized as the most common molecular abnormality in AML and FLT3ITD mutations are found in nearly 30% of AML patients. The poor prognosis of patients harboring FLT3 mutations renders FLT3 an obvious target of therapy. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3ITD positive and negative AML patients and as a maintenance therapy. So far, those clinical trials have showed very promising result. However, drug resistance to AC220 has also been reported through the early clinical studies. To understand the underlying mechanisms of drug resistance to AC220, we undertook an unbiased approach with a novel CRISPR pooled library to screen new genes whose loss of function confer resistance to AC220. In our screen, we identified SPRY3 and GSK3 as positive hits and the related Wnt and FGF-Ras-ERK signaling activation as the major mechanisms to cause resistance to the FLT3 inhibitor AC220. In the next step, we will explore the detailed underlying mechanisms by which SPRY3 and GSK3 mutations lead to drug resistance to AC220. We will also confirm our findings in primary AML patient samples. Taken together, our study will provide new insight into signaling pathways that contribute to the acquired resistance in AML. The knowledge learned from this study may lead to the development of more efficient therapeutic avenues for AML.

Citation Format: Jian Huang. A CRISPR/Cas9 knockout screen identify novel drug resistance genes in AML. [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 5042.
Abstract 5047: The MUC1 membrane-bound mucin increases tumor cell properties and chemoresistance in renal clear cell carcinoma
Authors: Gaudelot Kelly , Gnemmi Viviane , Bouillez Audrey , Gibier Jean-Baptiste , Fanchon Mélanie , Woszczyk Justine , Hémon Brigitte , Van Seuningen Isabelle , Aubert Sébastien , Perrais Michael
Published: July 2016

Introduction: MUC1, an O-glycoprotein membrane-bound mucin, is overexpressed in renal clear-cell carcinomas (CRCC) with correlation to two major prognostic factors, Tumor-Node-Metastasis stage and nuclear Fürhman grade. Previously, we have shown that (i) MUC1 was significantly overexpressed in metastatic CRCC vs non-metastatic CRCC and (ii) MUC1 is a target gene of HIF-1 transcription factor which is a part of the hypoxia pathway, the main renal carcinogenetic pathway. Furthermore, CRCC is highly resistant to common systemic chemotherapies.

Material and method: To better understand the roles of MUC1 in CRCC, we used two renal cell lines expressing MUC1 (786-O cells) or not (ACHN cells). 786-O cells were stably transfected with shRNA targeting MUC1 while ACHN cells with full-length MUC1. Proliferation, drug resistance, migration and invasion properties were studied in vitro in the different cellular clones using MTS cell proliferation assay, wound healing assay and Boyden chambers coated with Matrigel, respectively. Signaling pathways were screened by proteome profiler and western blot.

Results and discussion: We showed that MUC1 expression was associated with increased invasion and migration properties of renal carcinomatous cells and a decrease of cell-cell interactions. MUC1 overexpressing cells (i) expressed higher levels of anti-apoptotic factors and MDR genes involved in chemoresistance processes and (ii) were more resistant to chemotherapeutic drugs.

Conclusion: Our results show that MUC1 plays a role in biological properties of renal cancer cells suggesting important function for this mucin in tumour progression and chemo-resistance. Our data confirm its potential as a therapeutic target in this type of cancer.

Citation Format: Kelly Gaudelot, Viviane Gnemmi, Audrey Bouillez, Jean-Baptiste Gibier, Mélanie Fanchon, Justine Woszczyk, Brigitte Hémon, Isabelle Van Seuningen, Sébastien Aubert, Michael Perrais. The MUC1 membrane-bound mucin increases tumor cell properties and chemoresistance in renal clear 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 5047.
Abstract 5062: Hypoxia-induced amoeboid cancer cell migration
Authors: te Boekhorst Veronika , Jiang Liying , Lehmann Steffi , Zuidema Alba , Friedl Peter
Published: July 2016

Tumor hypoxia, by elevating hypoxia-inducible factors (HIFs), is an established inducer of epithelial-to-mesenchymal transition (EMT) and subsequent cancer cell invasion and metastasis. However, how hypoxia impacts on tumor cell invasion programs and translates into modes of dissemination of moving tumor cells remains unknown. Using breast cancer (BC) and head and neck cancer (HNC) cells which efficiently invade from multicellular spheroids into 3D fibrillar collagen, we addressed which signaling programs that control cell-cell adhesions, cell-matrix interactions and cytoskeletal dynamics and which modes of tumor cell migration are induced by hypoxia.

While both cancer models primarily invaded collectively under normoxic conditions, severe hypoxia (0.2% oxygen) or pharmacological stabilization of HIF-1 using the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) induced EMT-like detachment and migration of single cells into collagen, exhibiting a 2- and 4-fold increase of single BC and HNC cells, respectively. Besides infrequent spindle-shaped movement, hypoxia or DMOG predominantly induced the transition from collective to amoeboid single-cell migration, with blebby amoeboid migration as the predominant migration mode (4- and 3-fold increase for BC and HNC, respectively) next to filopodial amoeboid migration.

Hypoxia-induced amoeboid migration is characterized by variably fast and persistent migration, reaching up to several 100 μm/day, mediated by actin-rich filopodial or blebby protrusions at the leading edge. Criteria for amoeboid movement included (i) actin-containing blebs that interacted with collagen structures in a polarized fashion, (ii) weak dependence on matrix-metalloproteinase-mediated collagen remodeling, (iii) low dependence on focalized integrin-mediated matrix adhesions, and (iv) high dependence on RhoA-mediated actomyosin contraction for efficient bleb formation and migration speed. Interference with ROCK signaling using Y-27632 and myosin using blebbistatin significantly inhibited hypoxia-induced amoeboid dissemination and bleb formation. Thus, hypoxia induces plasticity of cancer invasion modes, with efficient blebby amoeboid dissemination as primary outcome. While blebby amoeboid motion is known as a rudimentary migration mode in embryonic stem cells, here we observe blebby amoeboid migration as a simple (low MMP- and integrin dependent) but efficient salvage migration mode of cancer cells under severe hypoxic stress.

Citation Format: Veronika te Boekhorst, Liying Jiang, Steffi Lehmann, Alba Zuidema, Peter Friedl. Hypoxia-induced amoeboid cancer cell migration. [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 5062.
Abstract 507: Prediction of colorectal cancer recurrence with plasma miRNAs in the ColoCare Study
Authors: Dickinson Brandon , Adams Scott V. , Ulrich Cornelia , Vickers Kathy , Wurscher Michelle , Grady William M. , Makar Karen W.
Published: July 2016

Background

Colorectal cancer is the third most common cancer and third most common cause of cancer-related death in the US. Recurrence of primary tumors occurs in 10-40% of patients depending on tumor stage. Early detection of recurrence can increase the treatment options for those eligible for curative therapy, which may lead to better survival. We determined the levels of multiple different plasma miRNAs and assessed their association with clinical outcomes to identify specific miRNAs that may serve as predictors of CRC recurrence.

Methods

Colorectal cancer patients (age 18-80, newly diagnosed, stage I-IV) were recruited as part of the ColoCare Study in Seattle, Washington, between 2007 and 2011. Patients who had a blood sample taken >30 and <365 days after surgery and prior to any recurrence were included in the study (n = 83).

Candidate miRNAs (miRNA 31, 141, 200b, 203, 16, 17-3p, 29a, 92a, 125b) were quantified using pre-designed TaqMan microRNA assays in triplicate assays. Plasma miRNA expression was normalized and expressed as copy number. Cox proportional hazards regression with robust standard errors was used to estimate hazard ratios of recurrence associated with level of each miRNA, adjusted for age, sex and tumor site and stratified by stage. No recurrences were observed in stage I patients. Statistical analyses were conducted separately for stages II-III and II-IV colorectal cancer.

Results

During a median of 1,067 days (33-2,254 days) follow-up after surgery, 20 recurrences were observed. In stage II-III patients, the presence of detectable circulating levels of miR-141 (HR = 4.30 [1.10-16.84]) or miR-203 (HR = 8.04 [1.39-46.4]) were associated with risk of recurrence. In stage II-IV patients, detectable miR-203 (HR = 3.52 [1.13-10.96]) was associated with recurrence, and elevated miR-29a levels were associated with greater risk of recurrence (HR: 1.80 per natural log unit, 95% CI: 1.06-3.04).

Conclusion

The present study identified multiple plasma miRNAs that may help to predict those patients at greatest risk of colorectal cancer recurrence. Our results require validation in order to determine the role for such plasma markers in clinical practice.

Citation Format: Brandon Dickinson, Scott V. Adams, Cornelia Ulrich, Kathy Vickers, Michelle Wurscher, William M. Grady, Karen W. Makar. Prediction of colorectal cancer recurrence with plasma miRNAs in the ColoCare Study. [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 507.
Abstract 5076: Ras association (RalGDS/AF-6) domain family member integrates with notch signaling to regulate tumor cell migration
Authors: Perumalsamy Lakshmi R. , S. Mahalingam
Published: July 2016

Tumor metastasis, the spread of tumor cells to distant tissues from its site of initiation is a multi-step and multi-factorial process. Hyperactivation of RAS pathway is reported in >50% of different cancers and is associated with metastasis and poor prognosis. Since Ras signaling pathway is essential for normal development, its been difficult to develop drugs against it in tumors. This led to the identification of several Ras effectors. Ras association (RalGDS/AF-6) domain family (RASSF) proteins are non-enzymatic effectors of Ras. RASSF7 is a member of the less characterized N-terminal RASSFs. Similar to other RASSFs, RASSF7 also associates with Ras. Several mutations within RASSF7 open reading frame as well as differential expression pattern were observed in various tumors. The role of RASSF7 and its downstream signaling is not yet clearly understood.

Microarray analysis for RASSF7 identified several differentially regulated genes with enrichment for pathways including cell adhesion, inflammation, actin cytoskeleton and cell-cell communication suggesting that RASSF7 might regulate cell migration. Ectopic expression of RASSF7 inhibited cell migration of multiple cancer cell lines in vitro. RT-qPCR and western blotting analysis suggest that RASSF7 can transcriptionally upregulate Notch1 expression. Knockdown of Notch1 abrogated RASSF7 mediated inhibition of cell migration. Our experiments suggest that RASSF7 alters the localization of Notch1. Cross-talk Notch signaling is a key signaling pathway regulating tumor progression across several different cancers. Collectively, our data suggests that RASSF7 impinges on Notch1 to regulate cell migration.

Citation Format: Lakshmi R. Perumalsamy, Mahalingam S. Ras association (RalGDS/AF-6) domain family member integrates with notch signaling to regulate tumor cell migration. [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 5076.
Abstract 5080: Amylase overexpression promotes ovarian cancer invasion
Authors: Mohamed Mai , Kruk Patricia
Published: July 2016

Introduction: Hyperamylasemia is an overexpression of amylase. Hyperamylasemia is associated with poor clinical outcome in ovarian cancer and reportedly serves as a prognostic biomarker for ovarian cancer. However, the function of hyperamylasemia in ovarian cancer is thus far unknown. Since amylase cleaves α1, 4-glycosidic bonds in carbohydrates, hyperamylasemia may play an active role in the etiology of ovarian cancer.

Purpose: The purpose of this study was to characterize amylase expression in ovarian cancer cell lines and establish an active role for hyperamylasemia in ovarian cancer.

Hypothesis: We hypothesized that secreted amylase enzymes cleave α1, 4-glycosidic bonds found in the carbohydrate shell which may result in a modified glycocalyx structure facilitating ovarian cancer cell invasion. Consequently, inhibiting amylase expression could abrogate ovarian cancer cell invasion/metastasis.

Methods: To characterize amylase isozyme expression, we assayed amylase expression in normal and cancerous ovarian epithelial cell lines by western immunoblot, qPCR, amylase assays and computational analyses. We measured the ability of amylase to promote cellular proliferation by MTS assays. We assayed invasive capacity of cultured cells using collagen coated Boyden chambers and abrogated amylase expression with either amylase siRNA or treatment with the blue-green algae, Spriulina.

Results: It was found that the amylase genes have identical or similar biochemical and bioinformatics properties: similar molecular weights, theoretical isoelectric points, disorder and hydropathy profiles, secondary structure and posttranslational modifications. In contrast to normal cell lines, ovarian cancer cell lines overexpress amylase at the messenger and protein levels and secrete high levels of metabolically active amylase. Amylase isozymes 1 and 2B were preferentially overexpressed in ovarian cancer cell lines. Inhibition of amylase abrogated ovarian cancer cell migration/invasion through collagen coated Boyden chambers which was associated with amylase-dependent alteration of extracellular carbohydrates. Further, the blue-green algae, Spirulina, was found to be a novel inhibitor of amylase.

Conclusion: Herein, we describe for the first time an active function of amylase to promote ovarian cancer cell invasion potentially mediated by amylase-dependent alteration of extracellular carbohydrates. Further, transcriptional inhibition of amylase by Spirulina demonstrates its clinical potential as an adjuvant nutraceutical therapy.

Citation Format: Mai Mohamed, Patricia Kruk. Amylase overexpression promotes ovarian cancer invasion. [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 5080.
Abstract 5086: Legumain-contained exosomes mediate remodeling of tumor extracellular matrix and protumor effects of TAMs
Authors: Shen Long , Shi Yuzhi , Xun Jing , Chen Chuanai , Du Lingfang , Tan Xiaoyue , Xiang Rong
Published: July 2016

It has been recognized that tumor associated macrophages (TAMs) in tumor microenvironment play a variety of pro-tumor effects during the tumor progression, while the role of extracellular matrix in mediating these effects is still unclear.

In this study, we focus on elucidating the way as well as the underlying mechanism about the involvement of an asparaginyl endopeptidase, legumain in the crosstalk between TAMs and ECM. Our data showed that co-culture of breast cancer cells 4T1 with macrophages Raw 264.7 promoted asparaginyl endopeptidase, legumain, expression with the introduction of markers of M2 - polarized macrophage. Legumain was detected in the condition medium (CM) collected from RAW 264.7 co-cultured with 4T1 cells by western blotting, with the existence of asparagine endopeptide enzyme activity assessed by enzyme activity assay. Result of western blotting showed that adding the CM of RAW 264.7 co-cultured with 4T1 cells depleted extracellular matrix (ECM) contents, fibronectin and collagen I. Transwell assay showed increased infiltration of 4T1 cells through the ECM contents after treatment with the CM of RAW 264.7 co-cultured with 4T1 cells. Furthermore, treatment with CM of RAW 264.7 co-cultured with 4T1 suppressed the adhesion of endothelial cells with ECM, promoting tube formation of endothelial cells compared with control. All the above results could be abolished by synthesized legumain inhibitor, RR-11a. Interestingly, legumain was also detected in the exosomes isolated from CM of RAW 264.7 co-cultured with 4T1. Applying legumain contained exosomes could replicate the effect of CM of RAW 264.7 co-cultured with 4T1 on the depletion of ECM contents, promotion of 4T1 infiltration and suppression of endothelial cell adhesion with ECM.

Altogether, our results suggest that legumain is overexpressed and secreted out by TAMs; TAMs exosomes contain legumain and help delivering legumain to the ECM thus exert its effect on the depletion of ECM, pro-infiltration and angiogenesis.

Citation Format: Long Shen, Yuzhi Shi, Jing Xun, Chuanai Chen, Lingfang Du, Xiaoyue Tan, Rong Xiang. Legumain-contained exosomes mediate remodeling of tumor extracellular matrix and protumor effects of TAMs. [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 5086.
Abstract 5095: Hic-5 modulation of the stromal matrix is required for mammary tumor progression
Authors: Goreczny Gregory J. , Ouderkirk Jessica , Olson Eric , Krendel Mira , Turner Christopher
Published: July 2016

Primary tumors grow and invade into surrounding tissues/stroma, frequently resulting in metastasis to distant organs. Tumor cell-stroma crosstalk plays a crucial, but incompletely understood role during tumor progression and metastasis. For example, increased stromal matrix density and/or rigidity correlates with poor prognosis in human breast cancer patients. Previous studies have implicated Hic-5, a focal adhesion scaffold protein, in tumor cell invasion, proliferation and metastasis. To investigate the role of Hic-5 in mammary tumor progression, Hic-5 -/- mice were generated and crossed with mice expressing Polyoma Middle T Antigen (PyMT) driven by the MMTV promoter. The PyMT mouse is a well-established model for human breast cancer progression. Tumors from the Hic-5 -/- PyMT mice demonstrated an increased latency and had reduced growth as compared to Hic-5 +/- PyMT controls. The absence of Hic-5 did not affect the progression of the tumor to a carcinoma stage, but there was a decrease in metastasis to the lungs. Immunohistochemical analysis showed that Hic-5 is primarily expressed in the cancer associated fibroblasts (CAFs) and the Hic-5 -/- PyMT tumor stroma exhibits reduced extracellular matrix (ECM) deposition. Furthermore, 3D cell-derived matrices (CDM) generated in vitro using isolated CAFs, confirmed that Hic-5 -/- PyMT CAFs do not efficiently deposit and organize ECM. The organization of the stromal matrix has been shown previously to increase the rigidity of the tumor, which in turn promotes FAK Y397 phosphorylation in the tumor cells to promote cell invasion and proliferation. Accordingly, the Hic-5 -/- PyMT tumors exhibited a reduction in FAK phospho-Y397 staining and attenuation of downstream ERK activation, suggesting that their stromal matrix is not optimally organized for tumor cell signaling. Taken together, these data demonstrate that Hic-5 expression in CAFs is important for stromal matrix organization/remodeling during tumor progression and that the loss of the organization of the ECM in the Hic-5 -/- tumor stroma may contribute to the reduced tumor growth and metastasis observed in these animals.

Supported by NIH CA163296 to CET, NS066071 to EO and Carol M. Baldwin Breast Cancer Fund to CET.

Citation Format: Gregory J. Goreczny, Jessica Ouderkirk, Eric Olson, Mira Krendel, Christopher Turner. Hic-5 modulation of the stromal matrix is required for mammary tumor progression. [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 5095.
Abstract 5096: Development of a decellularized tumor model for the evaluation of breast carcinomas
Authors: Martin Elizabeth , Pashos Nicholas , Gimble Jeffrey , Bunnell Bruce , Burow Matthew , Collins-Burow Bridgette
Published: July 2016

Breast cancer recurrence is a clinical manifestation of tumor progression in patients which has been shown to significantly increase mortality rates. While up to one-fifth of breast cancer patients will experience recurrence, the mechanisms underlying this process remain poorly understood. Recently, gene expression profiling of human tumor metastasis and recurrence demonstrated that the extracellular matrix (ECM)-receptor interactions pathway was enhanced. In breast carcinomas, the ECM is known to vary with cancer progression. The ECM acts as a chemical reservoir, structural component, and mediator of tumorigenesis to tumors. To date, the ECM of breast carcinomas has been evaluated within the context of a seeded cellular population, i.e. intact tumor. Here we demonstrate for the first time a mechanism to evaluate the ECM devoid of breast cancer cells. We have employed a model of tissue decellularization to breast cancer cell line derived tumors and demonstrate that tumor ECM architecture remains intact when devoid of cells. Validation of loss of cell content is evident through DNA quantification, nuclear DAPI stain, and H&E stain. Furthermore we demonstrate that structural proteins, fibronectin, collagen, and elastin are still retained within the decellularized scaffold. Applications of the tumor decellularization model will aid in the evaluation of ECM structural and molecular characteristics independent of cancer cell populations. The data presented here will enhance methods used to evaluate the cancer microenvironment and further our understanding of breast cancer recurrence, altering existing treatment strategies to extend survival.

Citation Format: Elizabeth Martin, Nicholas Pashos, Jeffrey Gimble, Bruce Bunnell, Matthew Burow, Bridgette Collins-Burow. Development of a decellularized tumor model for the evaluation of breast carcinomas. [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 5096.
Abstract 5097: Tissue stiffness regulates multinucleation in mammary epithelial cells
Authors: Simi Allison K. , Radisky Derek C. , Nelson Celeste M.
Published: July 2016

This study investigates how increased stiffness of the tumor microenvironment (TME) can facilitate cellular multinucleation.

Multinucleation is an easily observable marker for polyploidy, facilitating the quantification of this phenotype. Polyploidy is commonly an intermediate to aneuploidy, defined as an abnormal number of chromosomes, a phenotype found in 85% of solid cancers that can drive tumorigenesis. Up to 37% percent of tumors exhibit whole-genome doubling, which typically precedes other somatic copy number alterations. Additionally, induction of tetraploidy in human cells promoted increased tolerance for mutation, resistance to chemotherapeutic drugs, and transformation in culture.

Tumors are inherently stiffer than normal tissue, and this property has been shown to affect cell growth and proliferation. Similarly, cell cycle errors have long been linked to chromosomal abnormalities. Here, we used engineered two-dimensional substrata that mimic tumor and normal microenvironments to investigate how matrix stiffness regulates multinucleation in mammary epithelial cells.

Cells cultured on “stiff” substrata, representing tumor tissue, showed a nearly 14-fold increase in multinucleation compared to cells cultured on “soft” substrata, representing normal tissue. We found that multinucleation was regulated in part by signaling downstream of MMP3, a protease commonly upregulated in cancer. This signaling depended on expression of the Rac1 splice variant, Rac1b, production of reactive oxygen species (ROS), and expression of Snail. Under all conditions, cells cultured on soft substrata maintained a low frequency of multinucleation.

We also investigated biochemical effectors downstream of Snail. Aurora A kinase (Aurora A) is a mitotic regulator commonly elevated in cancer that has been shown to induce multinucleation in mammary epithelial cells and increase tumor incidence in mice. We found using microarray analysis that MMP3 increases the levels of Aurora A in mammary epithelial cells. We further discovered that upregulation of Aurora A in response to MMP3 signaling was limited to one transcript variant.

These data suggest that an increase in multinucleation is driven by the stiffening of the tissue during tumorigenesis, in part by MMP3-induced signaling and regulation of Aurora A splicing. More broadly, these data suggest a key role for the mechanical properties of the tumor microenvironment in cancer progression.

Citation Format: Allison K. Simi, Derek C. Radisky, Celeste M. Nelson. Tissue stiffness regulates multinucleation in mammary epithelial cells. [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 5097.
Abstract 5108: Glucagon like peptide 1 (GLP1) up-regulation in hypoxic and nutrient deprived microenvironment promotes neuroblastoma cell survival
Authors: Wesley Umadevi V. , Singh Nausheen , Dempsey Robert J.
Published: July 2016

Introduction: Glucagon-like peptide 1 (GLP1) an incretin has demonstrated to increase insulin secretion in a glucose-dependent manner and is shown to be neuroprotective. GLP1 also promotes angiogenesis. Hypoxia, a common feature in solid tumors including neuroblastoma, promotes tumor progression by upregulation of hypoxia responsive factors. Little is known about alterations of GLP1 expression in neuroblastoma under hypoxic condition. GLP1 is rapidly inactivated by dipeptidyl peptidase IV (DPPIV) mediated enzymatic cleavage. Previously we have shown that DPPIV suppresses neuroblastoma survival and angiogenesis. In this study, we examined whether GLP1, its receptor GLP1R, and DPPIV expressions are altered after exposing neuroblastoma cells to hypoxic conditions of oxygen and nutrient deprivation (OND). We then examined the direct effects of GLP1 on neuroblastoma proliferation and survival, and evaluated if DPPIV modulates GLP1 mediated functions.

Methods: We used Neuro-2a, a mouse neuroblastoma cells as a model. In vitro experiments were performed exposing these cells to conditions mimicking tumor areas that display oxygen and nutrient deprivation, followed by re-oxygenation. Immunofluorescence staining was used to analyze the protein expression of GLP1, GLP-1R, and DPPIV. RT-PCR was used to measure mRNA levels. Effects of DPPIV on GLP1 mediated Neuro-2a cell proliferation and survival was evaluated by MTT and live/dead cell assay. Levels of PI3K/AKT were determined by western blot analysis.

Results: Neuro-2a cells subjected to OND showed about 2 fold increase in the GLP1 protein and mRNA levels at four hours of

re-oxygenation after 4 hours of OND. However, GLP1 protein levels decreased drastically after one day of re-oxygenation, while DPPIV levels were highly upregulated. GLP1R was also downregulated at one day of re-oxygenation. At this time point, cell viability was decreased by 30-40%. Pre-treatment with DPPIV inhibitor rescued the protective effects of GLP1. Furthermore, exogenous GLP1 stimulated cell proliferation and increased Neuro-2a cell viability by 2-3 fold. GLP1dependent action was mediated via increased levels of phosphorylated AKT, a pro-survival signaling molecule. The effects of GLP1 were suppressed in presence of DPPIV.

Conclusion: The results indicate an inverse correlation between DPPIV and GLP1 levels in Neuro-2a cells. Together, these studies indicate that GLP1-DPPIV interaction may play an important role in promoting neuroblastoma cell proliferation and survival.

Citation Format: Umadevi V. Wesley, Nausheen Singh, Robert J. Dempsey. Glucagon like peptide 1 (GLP1) up-regulation in hypoxic and nutrient deprived microenvironment promotes neuroblastoma cell survival. [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 5108.
Abstract 5116: Phosphatidylserine-targeting antibodies augment anti-tumor activity of PD-1 antibodies and alter immuno-profiles in murine triple negative breast cancers
Authors: Gray Michael J. , Gong Jian , Parks Ryan N. , M.S. Hatch Michaela , Nguyen Van , Hughes Christopher C.W. , Hutchins Jeff T. , Freimark Bruce D.
Published: July 2016

Current treatments for triple negative breast cancers rely primarily upon surgical intervention and chemotherapy. Unfortunately, chemotherapies have the propensity to help establish an immunosuppressive condition in part though exposure of phosphatidylserine (PS) in the tumor microenvironment. We demonstrate that treatment of the murine triple negative breast cancer E0771 using a PS-targeting antibody inhibits in vivo growth, and significantly enhances the anti-tumor activity of antibodies directed to the checkpoint inhibitor PD-1. Combinational treatment by antibodies targeting PS and PD-1 increased the number of tumor infiltrating lymphocytes (TILs) to a greater extent than observed with single-arm therapies. Furthermore, combination of PS and PD-1 targeting antibodies provides a distinct survival advantage over treatment by either agent alone, which correlates with an increase in complete tumor regression and the onset of a durable immune mediated response capable of rejecting a secondary challenge by the same tumor. Finally, immune-profiling analysis of tumor RNA using Nanostring revealed that the combination antibody therapy enhanced immune regulatory pathways including MHC class I/II expression, antigen processing and presentation, and leukocyte and macrophage functions. Combined, our data suggest that in triple negative breast cancers antibody therapy blocking PS-associated immunosuppression may further enhance immunotherapies directed at immune checkpoint inhibitors, including those targeting the PDL-1/PD-1 axis.

Citation Format: Michael J. Gray, Jian Gong, Ryan N. Parks, Michaela M.S. Hatch, Van Nguyen, Christopher C.W. Hughes, Jeff T. Hutchins, Bruce D. Freimark. Phosphatidylserine-targeting antibodies augment anti-tumor activity of PD-1 antibodies and alter immuno-profiles in murine triple negative breast cancers. [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 5116.
Abstract 513: Standardization of technologies for CTC, ctDNA and miRNA enrichment, isolation and analysis for liquid biopsies during the first year of IMI's CANCER-ID
Authors: Schlange Thomas , Stoecklein Nikolas , Neves Rui P. , Pleier Sabrina , Bender Sebastian , Brychta Nora , Luetke-Eversloh Merlin V. , Andree Kiki , Terstappen Leon , Krahn Thomas , Krahn Thomas
Published: July 2016

Within the European Innovative Medicines Initiative (IMI) consortium CANCER-ID (www.cancer-id.eu), scientists at academic, clinical and industrial sites across Europe and in the US joined forces to evaluate innovative technologies in the field of liquid cancer biopsies. This project aims at implementing standard operating procedures (SOPs) for pre-analytical sample handling, enrichment, isolation and analysis of Circulating Tumor Cells (CTCs), circulating free tumor DNA (ctDNA) and microRNAs (miRNAs) as novel blood-based biomarkers, with a focus on Non-Small Cell Lung Cancer (NSCLC) and HER2-treatment refractory breast cancer. In order to determine sensitivity and specificity of different technologies for CTC isolation and analysis (e.g. detection of mutations, amplifications, protein phosphorylation), complex samples comprising a mixture of NSCLC or breast cancer cell lines spiked in healthy donor blood were distributed to different CANCER-ID partner sites. These cell lines have been selected based on their molecular/genetic properties to reflect clinically relevant subtypes of the disease and have been further characterized in terms of cell-surface marker expression and cell size distribution. The use of complex spiked samples better models the heterogeneity of real-life patient material. Furthermore, healthy donor and patient derived plasma samples are investigated using different technology platforms to validate tumor-specific miRNA or ctDNA profiles that might characterize molecular tumor subtypes. To this end, differences in exosome-derived versus free circulating miRNAs are of special interest. As for CTCs the development of ctDNA and miRNA standards that can be used to compare and validate different technologies are in the focus of this effort. In summary, our results pave the way for the next phase of CANCER-ID, which includes the analysis of cancer patient samples in clinical studies using different technologies and thereby advance the concept of liquid biopsy particularly in indications in which conventional tissue biopsies are difficult to obtain.

Citation Format: Thomas Schlange, Nikolas Stoecklein, Rui P. Neves, Sabrina Pleier, Sebastian Bender, Nora Brychta, Merlin V. Luetke-Eversloh, Kiki Andree, Leon Terstappen, Thomas Krahn, Thomas Krahn. Standardization of technologies for CTC, ctDNA and miRNA enrichment, isolation and analysis for liquid biopsies during the first year of IMI's CANCER-ID. [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 513.