bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4724: Combined clinical and biological risk stratification in pediatric hepatoblastoma
Authors: Cairo Stefano , Armengol Carolina , Häberle Beate , Simon-Coma Marina , Guettier Catherine , Leuschner Ivo , Buendia Marie-Annick , Schweinitz Dietrich von , Kappler Roland
Published: July 2017

Introduction Advances in chemotherapy and surgery have greatly improved overall survival rates of children with hepatoblastoma (HB). However, prognosis still remains quite poor for HB patients with high risk characteristics such as portal/hepatic venous macrovascular involvement, extrahepatic growth, high extent of the disease, and metastasis, as shown by studies of the International Childhood Liver Tumours Strategy Group (SIOPEL). The aim of our trinational study was to validate recently identified genetic and transcriptional characteristics of HB as prognostic biomarkers to aid development of a new risk stratification system based on clinical and biological factors.

Experimental procedures Tumor tissue of a cohort of 171 hepatoblastoma patients from Germany, France and Spain was analyzed for mutations in CTNNB1, NFE2L2 and TERT (Eichenmüller et al., J Hepatol 2014; 61:1312-1320) by Sanger sequencing and the 16-gene signature (Cairo et al., Cancer Cell 2008; 14:471-84) by real-time PCR. Kaplan-Meier estimates of specific survival time in the various groups were compared using the log-rank Mantel-Cox test.

Results Mutations of CTNNB1, NFE2L2, and TERT were found in 133 (78%), 10 (6%), and 10 (6%) patients, respectively. The adverse C2 subtype of the 16-gene signature was detected in 60 (35%) patients. Kaplan-Meier analyses depicted a significant association of the 16-gene signature (P<0.0001) and NFE2L2 mutations (P<0.0166) with poor outcome. Stratifying the patients of our cohort into a standard and high risk group based on the commonly used clinical SIOPEL criteria (Zsiros et al., J Clin Oncol 2010; 28:2584-2590) also revealed a strong association (P<0.0001) of the high risk group with poor outcome. When added to the SIOPEL risk groups, presence of at least one biomarker could discriminate the SIOPEL high risk patients into an intermediate and a very high risk group (P<0.0001), whereas biomarker positive and negative standard risk patients showed no difference in outcome.

Conclusions We have validated the 16-gene signature and NFE2L2 mutations as highly prognostic biomarkers in HB and propose a new stratification system that is based on the combination of clinical and biological factors, which might facilitate more tailored and risk-adapted therapies and thus better outcome of high risk patients in the future.

Citation Format: Stefano Cairo, Carolina Armengol, Beate Häberle, Marina Simon-Coma, Catherine Guettier, Ivo Leuschner, Marie-Annick Buendia, Dietrich von Schweinitz, Roland Kappler. Combined clinical and biological risk stratification in pediatric hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4724. doi:10.1158/1538-7445.AM2017-4724
Abstract 4741: Biomarker significance of plasma and tumor miR-21, miR-221, and miR-106a in pediatric osteosarcoma
Authors: Nakka Manjula , Allen-Rhoades Wendy , Li Yiting , Kelly Aaron , Shen Jianhe , Taylor Aaron , Barkauskas Donald A. , Yustein Jason , Andrulis Irene , Wunder Jay , Gorlick Richard , Meltzer Paul , Lau Ching , Man Tsz-Kwong
Published: July 2017

Osteosarcoma is the most common malignant bone tumor in children and young adults. Despite the use of surgery and multi-agent chemotherapy, osteosarcoma patients who respond poorly towards chemotherapy or develop relapses have a dismal outcome. Identification of biomarkers for active disease may help to monitor tumor burden, detect early relapses, and predict prognosis in these patients, so that they can be treated as early as possible to improve their survival. In this study, we examined if circulating microRNAs (miRNAs) can be used as biomarkers in osteosarcoma patients. We performed genome-wide miRNA profiling on a discovery cohort of OS plasma samples (n=32) collected from the Texas Children’s Hospital. A total of 56 miRNAs were upregulated and 164 miRNAs were downregulated in osteosarcoma samples when compared to normal plasma samples (n=8). miRNA-21, miR-221 and miRNA-106a were selected for further validation based on their known biological importance. We showed that all three circulating miRNAs were expressed significantly higher in OS samples (n=29) than normal samples (n=17) in an independent cohort obtained from the Children’s Oncology Group. Furthermore, we demonstrated that miR-21 was expressed significantly higher in osteosarcoma tumors (n=89) compared with normal bone controls. More importantly, lower expressions of miR-21 and miR-221, but not miR-106a, significantly correlated with a poor outcome. In conclusion, our results indicate that miR-21, miR-221 and miR-106a are elevated in the circulation of osteosarcoma patients, whereas tumor expressions of miR-21 and miR-221 are prognostically significant. Further investigation of the miRNAs may lead to a better prognostic method and potential miRNA therapeutics for osteosarcoma.

Citation Format: Manjula Nakka, Wendy Allen-Rhoades, Yiting Li, Aaron Kelly, Jianhe Shen, Aaron Taylor, Donald A. Barkauskas, Jason Yustein, Irene Andrulis, Jay Wunder, Richard Gorlick, Paul Meltzer, Ching Lau, Tsz-Kwong Man. Biomarker significance of plasma and tumor miR-21, miR-221, and miR-106a in pediatric osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4741. doi:10.1158/1538-7445.AM2017-4741
Abstract 4755: Calpastatin phosphorylation regulates radiation response in glioblastoma
Authors: Bassett Emily A. , Pearson Mitchell , Palanichamy Kamalakannan , Haque Saikh J. , Chakravarti Arnab
Published: July 2017

Glioblastoma (GBM) is the most prevalent and aggressive primary malignant brain tumor in adults. Patients diagnosed with GBM have a very poor prognosis and quality of life, with a median survival time of 12-15 months despite receiving the standard of care treatment. GBMs are highly heterogeneous, invasive, and resistant to both chemo- and radio-therapy. The overall goal of this study is to identify novel mechanisms of radioresistance in GBM, so that more effective therapies can be developed in the future. We performed quantitative mass spectrometry-based phosphoproteomic profiling of GBM cell lines before and after radiation treatment, and determined post-radiation fold-changes in phosphorylation for each phosphopeptide. Results show calpastatin as one of the statistically significant differentially regulated phosphopeptides following radiation treatment. Calpastatin is an endogenous inhibitor of calpains, which are calcium-dependent cysteine proteases that control a wide range of cellular processes including cell survival and apoptosis. We hypothesized that phosphorylation inactivates calpastatin resulting in increased calpain activity. Western blot analyses with phospho-specific calpastatin antibodies validated the mass spectrometry results. In order to test the functional significance of phosphorylated calpastatin, we utilized site-directed mutagenesis to generate non-phosphorylatable and phospho-mimetic calpastatin proteins. Western blot analyses of U87 cells stably expressing CAST-S633A or -S633E supported the hypothesis that calpastatin phosphorylation leads to increased calpain activity. In conclusion, we identified increased levels of calpastatin phosphorylation in multiple GBM cell lines following radiation treatment, and shown that this correlates with increased calpain activity. Modulation of calpastatin activity is a potential strategy to increase radiosensitivity of glioblastomas. Targeting calpastatin is advantageous over targeting calpain itself, since many calpain inhibitors exhibit limited selectivity. Results of this study will contribute to our understanding of how GBM tumors become resistant to radiation therapy. Funding: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01 (NCI), Brain Tumor Funders Collaborative Grant, and The Ohio State University CCC (all to AC).

Citation Format: Emily A. Bassett, Mitchell Pearson, Kamalakannan Palanichamy, Saikh J. Haque, Arnab Chakravarti. Calpastatin phosphorylation regulates radiation response in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4755. doi:10.1158/1538-7445.AM2017-4755
Abstract 4758: Sensitization of nasopharyngeal carcinoma to radiotherapy by targeting GFPT1/TGF-β1 signaling pathways
Authors: Chen Siqi , Gao Wei , Chan Jimmy Yu-Wai , Wong Thian-Sze
Published: July 2017

Background: Nasopharyngeal carcinoma (NPC) is a unique cancer with high susceptibility to radiation and mainstay treatment is radiotherapy. Effectiveness of NPC treatment is therefore dependent on the sensitivity of cancer cells to ionizing radiation treatment. Further, development of radio-resistant phenotype is a risk factor for distant metastasis and disease recurrence. Therefore, enhancing the degree of radiation sensitivity could improve prognosis and reduce the toxicity of radiotherapy with the use of lower radiation dose. Transforming growth factor β1(TGF-β1) expression is responsive to radiation treatment and the increase is important in the post-irradiation DNA repair. Tumor with low TGF-β1 level is more responsive to radiation treatment. Therefore, inhibiting TGF-β1 production can enhance the radiation damage to cancer cell DNA and also intensify the sensitivity of cancer cells to radiation. Our preliminary results indicate that TGF-β1 production in NPC is mediated by hexosamine pathway. Of which, GFPT1 (glucosamine-fructose-6-phosphate aminotransferase) is the rate-limiting enzyme.

Purpose of the study: To enhance radiation sensitivity by targeting GFPT1/ TGF-β1 cascade in NPC.

Experimental procedures: Radiation protective effects of TGF-β1 was examined by colony formation assay and Danio rerio (zebrafish) embryonic system. Production of TGF-β1 by activated hexosamine pathway in NPC was revealed by glucose and glucosamine treatment. Effects of GFPT1 silencing on radiosensitization are revealed by GFPT1 siRNA treatment followed by colony formation assay and gamma-H2AX formation assays.

Results: Pretreatment of HONE1 cells with recombinant TGF-β1 protein enhanced the clonogenic ability of irradiated cells. TGF-β1 significantly increased the survival of zebrafish embryos after irradiation. Glucose and glucosamine treatment increased the expression of TGF-β1 protein in HONE1 cells. Silence of GFPT1 reduced TGF-β1 mRNA and protein expression in HONE1 cells. Silencing GFPT1 reduced the clonogenic capacity of head and neck cancer cells after radiotherapy and enhanced the degree of radiation-induced DNA double-strand breaks.

Conclusions: Targeting GFPT1 enhances head and neck cancer cell sensitivity to radiation. GFPT1 inhibition can enhance the radiation sensitivity of head and neck cancer cells by inhibiting the expression of TGF-β1.

Citation Format: Siqi Chen, Wei Gao, Jimmy Yu-Wai Chan, Thian-Sze Wong. Sensitization of nasopharyngeal carcinoma to radiotherapy by targeting GFPT1/TGF-β1 signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4758. doi:10.1158/1538-7445.AM2017-4758
Abstract 4767: Identifying CDCA7 as a novel regulator of tumor-initiating cells in triple-negative breast cancer
Authors: Lin Chang-Ching , Lo Miao-Chia , Moody Rebecca , Stevers Nicholas , Tinsley Samantha , Wicha Max , Sun Duxin
Published: July 2017

Tumor-initiating cells (TICs) are a small fraction of cancer cells that stands at the apex of the tumor hierarchy. Given their tumor-initiating ability and resistance to conventional chemotherapies, TICs are considered potential therapeutic targets for preventing the relapse and metastasis of malignancies. Our group has identified a novel gene, Cell division cycle associated 7 (CDCA7), as an important regulator of TICs in triple-negative breast cancer (TNBC). CDCA7 is a target gene of MYC and NOTCH. Ectopic expression of CDCA7 has been demonstrated to induce neoplastic transformation in vitro and tumorigenesis in vivo. Despite its potential tumor-initiating ability, CDCA7 has not yet been well-studied in breast cancer. We found that CDCA7 was highly expressed in TNBC compared to other subtypes of breast cancer according to the RNA-sequencing data from The Cancer Genome Atlas. Furthermore, our Kaplan-Meier analysis of multiple datasets demonstrated that higher expression of CDCA7 was associated with poor prognosis in breast cancer. When we knocked down CDCA7 by siRNA in TNBC cell lines including SUM149, HCC1937, MDA-MB-231, and MDA-MB-436, CD44+/CD24- stem-like cell population and primary and secondary mammosphere formation were significantly decreased. In addition to suppressing the stem-like properties, knockdown of CDCA7 also inhibited the invasion and migration of breast cancer cells. These phenotypes indicated the inhibition of epithelial-to-mesenchymal transition (EMT), which was in accordance with the up-regulation of E-Cadherin and down-regulation of Vimentin after CDCA7 knockdown in TNBC cell lines. In conclusion, our results have demonstrated that CDCA7 is an important regulator of TICs in TNBC. Loss-of-function of CDCA7 leads to the inhibition of EMT and stemness in TNBC cells.

Citation Format: Chang-Ching Lin, Miao-Chia Lo, Rebecca Moody, Nicholas Stevers, Samantha Tinsley, Max Wicha, Duxin Sun. Identifying CDCA7 as a novel regulator of tumor-initiating cells in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4767. doi:10.1158/1538-7445.AM2017-4767
Abstract 4777: Cancer stemness and resistance: Napabucasin (BBI-608) sensitizes stemness-high cancer cells to Paclitaxel by inhibiting the STAT3-MUC1 pathway
Authors: Rogoff Harry A. , Li Juying , Li Chiang
Published: July 2017

Stemness-high cancer cells, or cancer stem cells (CSC) represent a subpopulation of cancer cells with enhanced tumorigenic capacity, metastasis-forming potential, and resistance to conventional chemotherapy and radiation. One such key CSC pathway is regulated by STAT3, a transcription factor that is downstream of several cytokines and growth factor receptors which controls the expression of a broad range of target genes and plays essential roles in CSC biology in many tumor types. Cancer stemness inhibitor Napabucasin (BBI-608), a small molecule that inhibits gene transcription driven by STAT3, can inhibit stemness gene expression, block spherogenesis, and kill CSCs. In vivo, BBI-608 effectively blocks cancer relapse and metastasis in xenograft models while sparing normal hematopoietic stem cells, suggesting that targeting stemness-high cancer cells is a feasible approach for developing next-generation cancer therapeutics to combat cancer recurrence.

SOX2 (sex-determining region Y-box protein 2), a transcription factor that is essential for self-renewal and pluripotency, is amplified in various cancer types and has been shown to be required for CSC self-renewal and maintenance. Mucin 1 (MUC1), a glycoprotein normally expressed at the apical surface of epithelial cells, is overexpressed in most human epithelial cancers. MUC1 has been implicated in regulating tumor proliferation, metabolism, invasion, angiogenesis, chemoresistance, and inflammation.

Here, we generated a stemness-high culture system based on the reporter activity of a SOX2-regulatory region construct in MKN28 gastric cancer cells (MKN28 SOX2-reporter GFP+ cells). BBI-608 treatment inhibited the STAT3-MUC1 pathway in stemness-high cells. High MUC1 status in stemness-high cells was associated with Paclitaxel resistance. Down-regulation of MUC1 sensitized stemness-high cells to Paclitaxel. Moreover, BBI-608 synergized with Paclitaxel in inhibiting spherogenesis of stemness-high cells. This study provides a new mechanism for the association of cancer stemness with drug resistance. Our findings support this combination therapy, which pairs a conventional chemotherapy (Paclitaxel) with a cancer stemness inhibitor (BBI-608), as a promising strategy to combat cancer.

Citation Format: Harry A. Rogoff, Juying Li, Chiang Li. Cancer stemness and resistance: Napabucasin (BBI-608) sensitizes stemness-high cancer cells to Paclitaxel by inhibiting the STAT3-MUC1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4777. doi:10.1158/1538-7445.AM2017-4777
Abstract 4788: Abraxane eliminates cancer stem cells in triple-negative breast cancers
Authors: Yuan Hebao , Guo Hongwei , Li Feng , Burnett Joseph , He Miao , Truchan Nathan , Myers Ila , Sun Duxin
Published: July 2017

Triple-negative breast cancer (TNBC) represents about 20 percent of all breast cancers, with a poorer survival rate and being associated with high-grade tumors and the least favorable prognosis. Due to the absence of hormone receptors and Her2 receptor, no targeted therapy is available and chemotherapy is the standard treatment for TNBC patient. Paclitaxel is a widely used chemotherapy drug for various cancers with severe side effects due to its solvent. Abraxane is an albumin-bound nanoparticle of paclitaxel with less severe side effects.

Here we investigated whether cancer stem cells (CSCs) in TNBC response differentially to abraxane and paclitaxel both in cell culture and xenograft mouse models.

Using a series of human TNBC cell lines, we showed that there is no difference between solvent-based paclitaxel and abraxane in vitro in term of efficacy and eliminating cancer stem cells using MTS assay, mammosphere formation assay, and flow analysis of cancer stem cells. Interestingly, we demonstrated that abraxane shows superior efficacy than solvent-based paclitaxel in human SUM149 xenograft NOD SCID mouse model in adjuvant treatment. More important, we demonstrated that abraxane eliminates cancer stem cells in xenograft NOD SCID mouse model in advanced treatment, while solvent-based paclitaxel increases cancer stem cells. Cell uptake assay indicated that TNBC cells take more abraxane than solvent-based paclitaxel, which partially attributes to its superior efficacy in xenograft mouse models. Taken together, our data confirmed that abraxane is superior to solvent-based paclitaxel and may be a better option for TNBC patients in adjuvant or metastatic treatment.

Citation Format: Hebao Yuan, Hongwei Guo, Feng Li, Joseph Burnett, Miao He, Nathan Truchan, Ila Myers, Duxin Sun. Abraxane eliminates cancer stem cells in triple-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4788. doi:10.1158/1538-7445.AM2017-4788
Abstract 4789: A novel MIF signaling pathway drives the malignant character of pancreatic cancer by targeting NR3C2
Authors: Yang Shouhui , He Peijun , Wang Liming , Wang Jian , Schetter Aaron , Tang Wei , Funamizu Naotake , Yanaga Katsuhiko , Uwagawa Tadashi , Satoskar Abhay R. , Gaedcke Jochen , Bernhardt Markus , Ghadimi B. Michael , Gaida Matthias M , Bergmann Frank , Werner Jens , Ried Thomas , Hanna Nader , Alexander H. Richard , Hussain S. Perwez
Published: July 2017

Pancreatic cancers with aberrant expression of macrophage migration inhibitory factor (MIF) are particularly aggressive. To identify key signaling pathways that drive disease aggressiveness in tumors with high MIF expression, we analyzed the expression of coding and noncoding genes in high and low MIF-expressing tumors in multiple cohorts of pancreatic ductal adenocarcinoma (PDAC) patients. The key genes and pathways identified were linked to patient survival and were mechanistically, functionally, and clinically characterized using cell lines, a genetically engineered mouse model, and PDAC patient cohorts. Here, we report evidence of a novel MIF-driven signaling pathway that inhibits the orphan nuclear receptor NR3C2, a previously undescribed tumor suppressor that impacts aggressiveness and survival in PDAC. Mechanistically, MIF upregulated miR-301b that targeted NR3C2 and suppressed its expression. PDAC tumors expressing high levels of MIF displayed elevated levels of miR-301b and reduced levels of NR3C2. In addition, reduced levels of NR3C2 expression correlated with poorer survival in multiple independent cohorts of PDAC patients. Functional analysis showed that NR3C2 inhibited epithelial-to-mesenchymal transition and enhanced sensitivity to the gemcitabine, a chemotherapeutic drug used in PDAC standard of care. Furthermore, genetic deletion of MIF disrupted a MIF-mir-301b-NR3C2 signaling axis, reducing metastasis and prolonging survival in a genetically engineered mouse model of PDAC. Taken together, our results offer a preclinical proof of principle for candidate therapies to target a newly described MIF-miR-301b-NR3C2 signaling axis for PDAC management.

Citation Format: Shouhui Yang, Peijun He, Liming Wang, Jian Wang, Aaron Schetter, Wei Tang, Naotake Funamizu, Katsuhiko Yanaga, Tadashi Uwagawa, Abhay R. Satoskar, Jochen Gaedcke, Markus Bernhardt, B. Michael Ghadimi, Matthias M Gaida, Frank Bergmann, Jens Werner, Thomas Ried, Nader Hanna, H. Richard Alexander, S. Perwez Hussain. A novel MIF signaling pathway drives the malignant character of pancreatic cancer by targeting NR3C2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4789. doi:10.1158/1538-7445.AM2017-4789
Abstract 4808: Effect of Merkel cell polyomavirus large and small T-antigen on the thrombospondin and the periostin promoter
Authors: Konstantinell Aelita , Furuholmen Ida Sofie , Sveinbjørnsson Baldur , Moens Ugo
Published: July 2017

Merkel cell carcinoma (MCC) is a rare, but aggressive form of skin cancer with rising incidence and high mortality rate. Approximately 80% of MCC tumors are positive for Merkel cell polyomavirus (MCPyV), suggesting a causative relationship between MCPyV and MCC. Exosomes are 30-150 nm vesicles that contain proteins, lipids, mRNAs, lncRNAs, and miRNAs. Exosomes from virus-infected cells comprise also viral nucleic sequences and proteins. Because tumor cell-derived exosomes may contribute to cancer, we hypothesized that MCPyV may affect the composition of exosomes and play a role in tumorigenesis. Therefore, we compared the proteins in exosomes produced by MCPyV-positive MKL1 and MKL2 and MCPyV-negative MCC13 and MCC26 MCC cell lines.

Our proteomic analysis of exosomes originating from polyomavirus-negative and polyomavirus-positive MCC cell lines revealed the presence of the oncogenic proteins periostin and thrombospondin. Western blot analysis of exosomes and lysates of these MCC cells confirmed the presence of these proteins in exosomes from all cell lines. Thrombospondin, but not periostin was detectable in cell lysates, and an enrichment of both proteins was detected in exosomes of all cell lines. The effect of MCPyV large T-antigen (LT-ag) and small t-antigen (st-ag) on the periostin and thrombospondin promoters was examined by transient transfection studies with luciferase reporter plasmids. Transfection experiments in MCC13 revealed that LT-ag, but not st-ag significantly increased the activity of the thrombospondin and periostin promoters. In MCC26 cells, neither LT-ag nor st-ag had a significant effect on the thrombospondin promoter activity, while both proteins alone or in combination significantly stimulated the periostin promoter strength. Our results suggest that MCPyV proteins may contribute to tumorigenesis by enhancing the expression of the oncoproteins thrombospondin and periostin and promote their secretion via exosomes.

Citation Format: Aelita Konstantinell, Ida Sofie Furuholmen, Baldur Sveinbjørnsson, Ugo Moens. Effect of Merkel cell polyomavirus large and small T-antigen on the thrombospondin and the periostin promoter [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4808. doi:10.1158/1538-7445.AM2017-4808
Abstract 4834: 3D modeling of immune cell interactions in breast cancer and prediction of immunotherapy response
Authors: Guo Qi , Shuford Stephen , McKinley Brian , Rippon Mary , Cornett Wendy , O'Rourke Mark , Schammel David , Edenfield Jeff , Kaplan David L. , Crosswell Hal E. , Desrochers Teresa
Published: July 2017

While breast cancer has an overall 5-year survival rate of 89%, the rate for patients with stage 4 metastatic disease is only 26%. Immunotherapies have the potential to improve the prognosis for these patients while also providing better treatment options for all breast cancer patients since they have fewer side effects enabling longer treatment times and the use of combination therapies and reduced chances of developing resistance. Currently these treatments are tested in standard 2D cell cultures that are inaccurate in mimicking in vivo drug response or animal models where the immune system differs from humans in numerous ways including T-cell subsets, cytokine receptors, and costimulatory molecule expression. We have developed 3D models of human breast cancer that span the subtypes, ER+, HER2+, and triple negative, incorporate numerous stromal cell types, fibroblasts and adipocytes, and include different immune cells, macrophages and T-cells under either static or perfusion culture systems. These models have been used to examine how tumor cells influence macrophage differentiation using undifferentiated peripheral blood mononuclear cells (PBMCs), how M1 and M2 macrophages influence tumor cell survival and proliferation, how the combination of these cell types influence cytokine secretion, and how the microenvironment affects macrophage invasion. We have also used these complex models to examine response of tumor cells and T-cells to checkpoint inhibitors through standard viability assays and flow cytometry. These models have several potential uses which include the ability to quickly answer whether a particular immunotherapy agent is effective for that particular patient-specific manner and to screen potential novel immunotherapeutic candidates and/or combinations prior to clinical use.

Citation Format: Qi Guo, Stephen Shuford, Brian McKinley, Mary Rippon, Wendy Cornett, Mark O'Rourke, David Schammel, Jeff Edenfield, David L. Kaplan, Hal E. Crosswell, Teresa Desrochers. 3D modeling of immune cell interactions in breast cancer and prediction of immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4834. doi:10.1158/1538-7445.AM2017-4834
Abstract 4847: NME1 mediates a switch in beta integrin subunits that correlates with prolonged patient survival
Authors: Leonard M. Kathryn , Snow Grace , Novak Marián , McCorkle Joseph R. , Yang Xiuwei H. , Belkin Alexey , Kaetzel David M.
Published: July 2017

Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility, invasion, and metastasis, but the molecular mechanisms underlying this activity are not completely understood. Herein we report a novel mechanism through which NME1 modulates focal adhesion dynamics via regulation of integrins β1 and β3. Stable expression of NME1 significantly altered focal adhesion turnover at the cell periphery. Interestingly, over-expression of NME1 resulted in a switch from predominantly fast recycling α4β1 integrins to slower recycling αvβ3 integrins. Contrary to its regulation of other cell surface receptors, the inhibition of integrin β1 and induction of integrin β3 by NME1 was found to occur at the transcriptional level rather than through dynamin mediated endocytosis. Induction of integrin β3 required both the 3-5’ exonuclease and nucleoside diphosphate kinase (NDPK) activities of NME1, which are also required for its metastasis suppressor activities in vivo. Further suggesting that the induction of integrin β3 is involved in the metastasis suppression function of NME1, knockdown of integrin β3 significantly increased the invasion capability of cells expressing NME1 in vitro compared to control cells. Analysis of metastatic melanoma patients in the TCGA showed individuals with a 1.5x or greater increase in integrin β3 mRNA had a significantly longer overall survival times. Additionally, an inverse correlation was observed between NME1 and integrin β1 mRNA in an independent microarray of primary melanomas. The inverse correlation of NME1 and integrin β1 RNA was also a strong predictor of prolonged distant disease free and overall survival in patients with the basal-like subtype of breast carcinoma. Together, these data strongly suggest NME1 prevents metastasis of human melanoma and some types of breast cancers by altering beta integrin expression to reduce recycling of focal adhesions and, ultimately suppress of cell motility.

Citation Format: M. Kathryn Leonard, Grace Snow, Marián Novak, Joseph R. McCorkle, Xiuwei H. Yang, Alexey Belkin, David M. Kaetzel. NME1 mediates a switch in beta integrin subunits that correlates with prolonged patient survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4847. doi:10.1158/1538-7445.AM2017-4847
Abstract 4854: Evaluating HDAC6 as a causal factor in metastatic breast cancer to develop immunotherapy
Authors: Banik Debarati , Hadley Melissa , Kim Jennifer , Knox Tessa , Nair Jayakumar , Kozikowski Alan , Shen Sida , Vyas Charu , Donohue Ashleyn , Sotomayor Eduardo , Villagra Alejandro
Published: July 2017

Histone deacetylases (HDACs), originally described as histone modifiers, have recently been demonstrated to modify a variety of other proteins involved in diverse cellular processes unrelated to the chromatin environment, including the modulation of proteins related to cell cycle/apoptosis and immune regulation. In contrast to the well-documented effects of HDAC inhibitors (HDACi) in the control of cell cycle and apoptosis, their role in immune-biology is incompletely understood. We have found that the pharmacological or genetic abrogation of a single HDAC, i.e. HDAC6, inactivates the STAT3 pathway and modulates the expression of immuno-regulatory proteins, including the down-regulation of PD-L1, PD-L2 and B7-H4, important negative regulators of immune function, often over-expressed in cancer cells; including breast cancer. HDAC6 has been also involved in a number of structural functions related to cellular motility, shape and intracellular transport through the regulation of the acetylation of numerous targets, including tubulin and cortactin. This function is strongly suggestive of HDAC6 being a key player in metastatic cancer progression. In our initial studies we observed that the selective HDAC6 inhibitor Nexturastat A is capable of reducing the tumor growth in a highly aggressive murine mammary carcinoma that mimics human triple negative breast cancer (TNBC), under both orthotopic and subcutaneous conditions of implantation. Additionally, we observed that the size and number of secondary tumor nodules in lungs were significantly diminished after the HDAC6i treatment. In order to boost the anti-tumor T-cell response, we also tested check-point inhibitors against the tumor (such as anti PD-1 and CTLA4 antibodies). While each of the standalone treatments showed a certain degree of success in reducing tumor growth and enhancing intra-tumoral IFNγ, we demonstrated that HDAC6i improves anti-tumor immune responses when combined with immune check-point blockade.

Citation Format: Debarati Banik, Melissa Hadley, Jennifer Kim, Tessa Knox, Jayakumar Nair, Alan Kozikowski, Sida Shen, Charu Vyas, Ashleyn Donohue, Eduardo Sotomayor, Alejandro Villagra. Evaluating HDAC6 as a causal factor in metastatic breast cancer to develop immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4854. doi:10.1158/1538-7445.AM2017-4854
Abstract 4857: TAK1 mediated IL1 expression as autocrine signaling to promote breast cancer metastasis
Authors: Iriondo Oihana , Elhodaky Mostafa , Liu Yarong , Lee Grace , Lang Julie E. , Wang Pin , Yu Min
Published: July 2017

TGFβ-activated kinase 1 (TAK1), a serine/threonine kinase from the MAPKKK family, is regulated by different cytokines such as IL1, TGFβ, TNFα and BMPs, and it is therefore a key player in the cellular responses induced by changes in the microenvironment. TAK1 regulates cell survival, differentiation and inflammatory responses by activating other intracellular kinases such as p38, JNK or IKK. TAK1 activation is thought to influence the progression of several types of cancer, including lymphomas and pancreatic, colon, liver and breast cancer. In this study, we aimed to investigate the mechanisms by which TAK1 regulates breast cancer progression. (5Z)-7-Oxozeanol is a compound that inhibits the kinase activity of TAK1. Treatment of mice injected with MDA-MB-231 cells with (5Z)-7-Oxozeanol containing nanoparticles reduced metastasis formation, while growth of the primary tumor was not affected. TAK1 involvement in metastasis was confirmed by xenotransplantation experiments done with MDA-MB-231 cells overexpressing wild type or dominant negative forms of TAK1. Using these stable cell lines, we confirmed that TAK1 is required for p38 activation induced by several cytokines. Interestingly, cells cocultured with macrophages showed a TAK1-dependent increase in the expression levels of IL1α and IL1β, which can lead to TAK1 activation themselves. These cytokines were also detected in cancer cells that colonized the mouse lung, suggesting that the lung microenvironment could promote the initiation of a positive feedback loop by cancer cells, promoting the growth of metastatic lesions. Disruption of this positive autocrine feedback loop may be a potential therapeutic approach to suppress breast cancer metastasis to the lung.

Citation Format: Oihana Iriondo, Mostafa Elhodaky, Yarong Liu, Grace Lee, Julie E. Lang, Pin Wang, Min Yu. TAK1 mediated IL1 expression as autocrine signaling to promote breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4857. doi:10.1158/1538-7445.AM2017-4857
Abstract 4861: The role of PRAME in promoting uveal melanoma metastasis
Authors: Sanchez Margaret I. , Field Matthew G. , Kuznetsov Jeffim N. , Kurtenbach Stefan , Pham Dien , Harbour James W.
Published: July 2017

Uveal melanoma (UM) is the most common primary eye cancer and has a predisposition for fatal metastasis. Our group has shown that UMs can be categorized by gene expression profiling (GEP) into two molecular classes associated with metastatic risk: Class 1 (low metastatic risk) and Class 2 (high metastatic risk). While the majority of Class 2 UMs (71%) metastasize, only 38% of Class 1 UMs metastasize. Our group was the first to identify the cancer-testis antigen PRAME as a biomarker for increased metastatic risk in Class 1 UMs, and sub-categorized these tumors into Class1 PRAME+ (intermediate metastatic risk) and Class1 PRAME- (low metastatic risk) subgroups. Further we identified PRAME+ was associated with shorter time to metastasis and worse prognosis in class 2 patients. We have now shown that this PRAME expression is correlated with hypomethylation in the promoter region of the gene, suggesting a mechanism for its expression. Since we have previously shown that PRAME is associated with early metastasis in Class 1 and Class 2 UMs, we hypothesized that PRAME plays a direct role in UM metastasis. To better understand the role of PRAME in UM, we used Class1PRAME+ (F41) and Class1PRAME– (Mel290) UM cell lines to develop in vitro models to study the role of PRAME. Since F41 cells are PRAME+ and highly metastatic, we constitutively silenced PRAME with a V5 tag (F41 shPRAME-V5). For Mel290 cells, we overexpressed PRAME to see whether this cell line, which does not induce metastatic death, became more malignant. We confirmed the silencing and overexpression of PRAME in our transformed cells with western blot. For cell proliferation, we found a slight increase in the Mel290 PRAME+ cells compared to non-transformed Mel290. Strikingly, in F41 cells, knockdown of PRAME led to complete cell death. In a xenograft animal model, NSG male mice were injected with either Mel290 PRAME-V5, Mel290, F41 shPRAME-V5 or F41 TET-empty vector cells. We found that PRAME overexpressing cell lines (Mel290 PRAME-V5 and F41 TET-empty vector) induced rapid, multiple and aggressive liver metastases that led to death, whereas the non-expressing PRAME cells (Mel290 and F41 shPRAME-V5) did not present macro or micrometastasis in any cases. This data highly suggests that PRAME overexpression is involved in UM metastasis. Thus, PRAME mRNA expression is not only a prognostic biomarker but also could be an important target for treatment.

Citation Format: Margaret I. Sanchez, Matthew G. Field, Jeffim N. Kuznetsov, Stefan Kurtenbach, Dien Pham, James W. Harbour. The role of PRAME in promoting uveal melanoma metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4861. doi:10.1158/1538-7445.AM2017-4861
Abstract 4866: CD47 promotes metastasis and proliferation of colorectal cancer via MAPK/ERK pathway
Authors: Hu Tuo , Chen Yu-feng , Zhou Chi , Ke Jia , Liu Hua-shan , Zheng Xiao-bin , Wang Jian-ping , Wu Xiao-jian , Wu Xian-rui , Lan Ping
Published: July 2017

Background and Aim: A number of studies demonstrated that CD47 was overexpressed in several kinds of tumors including colorectal cancer (CRC). Furthermore, the level of CD47 was positively correlated with tumor proliferation, stemness and metastasis. The aim of this study is to evaluate the role of CD47 in the metastasis and proliferation in CRC.

Methods: We used the data from GEO database GSE8671 to analyze the expression level of CD47 and its relationship with prognosis in CRC. The expression levels of CD47 in normal and CRC tissues from our hospital were detected using quantitative RT-PCR and western blot. CD47 was downregulated or overexpressed via lentiviral vector system. Then the RTCA assay and migration assay were carried out to investigate the function of CD47 in cell proliferation and migration. Western blot for cancer related pathways was performed to find out the downstream signaling pathways of CD47.

Results: Increased expression of CD47 was found in CRC tissues. Inhibition of CD47 expression in HCT116 by ShRNA attenuated the capacity of cell proliferation and migration. On the other hand, overexpression of CD47 in DLD1 increased the capacity for cell proliferation and migration. CD47 induced cell proliferation and migration was found to be associated with the activation of MAPK/ERK pathway.

Conclusions: High expression of CD47 was found to be associated with the proliferation and metastasis in CRC via the activation of MAPK/ERK signaling pathway. These findings provide an SIRPα-independent mechanism for CD47 in the progression of CRC, which may find a novel therapy target for the precise treatment of CRC.

Citation Format: Tuo Hu, Yu-feng Chen, Chi Zhou, Jia Ke, Hua-shan Liu, Xiao-bin Zheng, Jian-ping Wang, Xiao-jian Wu, Xian-rui Wu, Ping Lan. CD47 promotes metastasis and proliferation of colorectal cancer via MAPK/ERK pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4866. doi:10.1158/1538-7445.AM2017-4866
Abstract 4868: RD3 reprogramming in N-MYC non-amplified neuroblastoma evolution
Authors: Somasundaram Dinesh Babu , Subramanian Karthikeyan , Aravindan Sheeja , Herman Terence , Natarajan Aravindan
Published: July 2017

N-MYC amplification is restricted to ~30% of the high-risk neuroblastoma (NB), while the remaining 70% is N-MYC non-amplified (N-MYC-NA), yet still has a poor outcome with only 37% 5 Year OS and a miserable 9% 10Y OS. Recently, we demonstrated, Retinal Degeneration Protein 3 (RD3) regulates metastatic state of NB cells and its loss associates with poor prognosis. Herein, utilizing bed-to-bench approach coupled with in vitro, in vivo and ex vivo NB models, we investigated the role of RD3 reprogramming in the evolution of N-MYC-NA-NB. Assessing 15 different human derived stage-4 N-MYC-NA-NB cell lines demonstrated: transcriptional/translational loss of RD3 compared to neural crest cells; cell specific loss and; association of RD3 loss to cellular progressive disease, cellular therapy resistance and disease relapse. Further, in silico analysis demonstrated that RD3-loss was intrinsically associated with reduced OS and abridged relapse-free survival in N-MYC-NA-NB patient cohorts. We also demonstrated the complete loss of RD3 in metastatic site-derived aggressive cells (regardless of CSC status) ex vivo and in reproducible aggressive disease models in vivo. RD3-loss correlated with the heightened metastatic state (tumor cell migration, invasion and tumorosphere formation) of the N-MYC-NA-NB cells. Further, re-expressing RD3 in aggressive cells reverted their metastatic potential both in vitro and in vivo. These results demonstrate the loss of RD3 in high-risk N-MYC-NA-NB, its novel tumor evolution stabilization function and further imply that continuous ongoing acquisition of RD3-loss in therapy resistant cells may directly relate to tumor progression and poor clinical outcomes.

Citation Format: Dinesh Babu Somasundaram, Karthikeyan Subramanian, Sheeja Aravindan, Terence Herman, Aravindan Natarajan. RD3 reprogramming in N-MYC non-amplified neuroblastoma evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4868. doi:10.1158/1538-7445.AM2017-4868
Abstract 4919: Ablation of neuropilin 1 from glioma-associated microglia and macrophages slows tumor progression
Authors: Miyauchi Jeremy T. , Chen Danling , Choi Matthew K. , Shroyer Kenneth , Selwood David , Tsirka Stella E.
Published: July 2017

Gliomas are the most commonly diagnosed primary tumors of the central nervous system (CNS). Median times of survival are dismal regardless of the treatment approach, underlying the need to develop more effective therapies. Modulation of the immune system is a promising strategy as innate and adaptive immunity play important roles in cancer progression. Glioma associated microglia and macrophages (GAMs) can comprise over 30% of the cells in glioma biopsies. Gliomas secrete cytokines that suppress the anti-tumorigenic properties of GAMs, causing them to secrete factors that support the tumor’s spread and growth. Neuropilin 1 (Nrp1) is a transmembrane receptor that in mice both amplifies pro-angiogenic signaling in the tumor microenvironment and affects behavior of innate immune cells. Using a Cre-lox system, we generated mice that lack expression of Nrp1 in GAMs. We demonstrate, using an in vivo orthotopic glioma model, that tumors in mice with Nrp1-deficient GAMs exhibit less vascularity, grow at a slower pace, and are populated by increased numbers of anti-tumorigenic GAMs. Moreover, glioma survival times in mice with Nrp1-deficient GAMs were significantly longer. Treating wild-type mice with a small molecule inhibitor of Nrp1’s b1 domain, EG00229, which we show here is selective for Nrp1 over Nrp2, yielded an identical outcome. Moreover, transplanting bone marrow from mice with Nrp1-deficient macrophages to wild type recipient mice led to reductions in tumor growth, al-be-it not to the extent seen in knockout mice. Nrp1-deficient or EG00229-treated wild-type microglia exhibited a shift towards anti-tumorigenicity as evident by altered inflammatory marker profiles in vivo and decreased SMAD2/3 activation when conditioned in the presence of glioma-derived factors. These results provide support for the proposal that pharmacological inhibition of Nrp1 constitutes a potential strategy for suppressing glioma progression.

Citation Format: Jeremy T. Miyauchi, Danling Chen, Matthew K. Choi, Kenneth Shroyer, David Selwood, Stella E. Tsirka. Ablation of neuropilin 1 from glioma-associated microglia and macrophages slows tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4919. doi:10.1158/1538-7445.AM2017-4919