bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 215: Tracking transplanted cells with paramagnetic fluorinated nanoemulsions
Authors: Kislukhin Alexander A. , Xu Hongyan , Adams Stephen R. , Narsinh Kazim , Tsien Roger Y. , Ahrens Eric T.
Published: August 2015

Visualization of distinct cell populations in vivo is one of the most formidable challenges in biomedical sciences. Clinical translation of cutting-edge therapies that involve administration of live immunotherapeutic or stem cells can benefit from understanding the fate of injected cells in vivo. Magnetic resonance imaging (MRI) is emerging as a prime method for non-invasive, longitudinal cell tracking. Fluorine-19 MRI (19F MRI) involves the use of non-radioactive 19F label in the form of biologically inert, cytocompatible perfluorocarbons (PFC). Immediately prior to injection, cells of interest are labeled ex vivo with water-based PFC nanoemulsions. 19F MRI yields background-free images of labeled cells in an anatomical context provided by conventional 1H MRI. Both datasets are acquired on the same scanner in one imaging session. This technology has recently been used to detect immunotherapeutic dendritic cells delivered to colorectal adenocarcinoma patients.

We sought to enhance sensitivity and versatility of 19F MRI by using paramagnetic relaxation agents. Covalent modification of clinically relevant PFCs with metal-binding ligands enabled stable and uniform incorporation of gadolinium and iron in the fluorous phase of nanoemulsions. Paramagnetic relaxation enhancement resulted in up to 50-fold reduction in 19F longitudinal relaxation time (T1) compared to state-of-the-art commercial tracers. The ultralow 19F T1 of our paramagnetic tracers enabled in vivo cell tracking with rapid, three-dimensional, zero time-to-echo (ZTE) 19F MRI pulse sequences in mice bearing subcutaneous allografts of 19F-labeled GL261 cells. Moreover, the high local concentration of paramagnetic metals imparted negative contrast in T2*-weighted 1H images.

Combination of the new agents with conventional 19F tracers comprises a previously unavailable toolbox of 19F contrast agents, selectively detectable by appropriate acquisition methods. This enables “multicolor” 19F MRI tracking of multiple cell populations, e.g., for monitoring the interaction of host immune cells with injected therapeutic cells or cancer xenografts. Overall, the design of biocompatible, fast-relaxing 19F tracers addresses the issue of modest sensitivity of 19F MRI and reduces the barriers to widespread adoption of this powerful imaging technique.

Citation Format: Alexander A. Kislukhin, Hongyan Xu, Stephen R. Adams, Kazim Narsinh, Roger Y. Tsien, Eric T. Ahrens. Tracking transplanted cells with paramagnetic fluorinated nanoemulsions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 215. doi:10.1158/1538-7445.AM2015-215
Abstract 2153: Kinome inhibitor screen identifies kinase inhibitors that inhibit selectively the survival of mutant K-Ras-dependent human cancer cells
Authors: Berndt Norbert , Zhang Xiaolei , Jiang Rays H.Y. , Sebti Said M.
Published: August 2015

While mutant (mt) KRas has proven to be difficult to target directly, mt KRas-driven cancer cells may gain dependencies on other signal transduction pathways. Therefore, exploring these dependencies may lead to the discovery of pathways and targets whose inhibition may induce synthetic lethality in cancer cells that depend on mt KRas. We have screened a kinase inhibitor library against 7 human cancer cell lines that harbor mt KRas; 4 mt KRas-dependent (A427 and H358 (lung), SW620 and SK-CO-1 (colon)); and 3 mt KRas-independent (HCT116, DLD-1 and HCT-8 (colon)) to identify kinase inhibitors that selectively inhibit the survival of mt KRas-dependent cancer cells. Our results show that 13 of the kinase inhibitors suppressed potently the survival of all 7 human cancer cell lines, 31 inhibited very weakly and 84 inhibited differentially each cell line, and the remaining compounds showed varied activities across cell lines. Importantly, 7 kinase inhibitors inhibited preferentially (p< 0.01) the viability of mt KRas-dependent over -independent human cancer cell lines. These results suggested that the 7 compounds may target kinases whose inhibition preferentially affects mt KRas-dependent cells. Analysis of the kinases that are targeted by these inhibitors revealed that these compounds target predominantly tyrosine kinases suggesting that at least in these cell lines these compounds may disrupt mt KRas “addiction” by inhibiting tyrosine kinase-dependent signaling.

Citation Format: Norbert Berndt, Xiaolei Zhang, Rays H.Y. Jiang, Said M. Sebti. Kinome inhibitor screen identifies kinase inhibitors that inhibit selectively the survival of mutant K-Ras-dependent human cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2153. doi:10.1158/1538-7445.AM2015-2153
Abstract 216: Pancreatic cancer fluorescence-­guided surgery with a fluorophore­-conjugated antibody to carcinoembryonic antigen (CEA) improves surgical resection and increases disease-free and overall survival in orthotopic mouse models
Authors: Metildi Cristina A. , Kaushal Sharmeela , Luiken George A. , Hoffman Robert M. , Bouvet Michael
Published: August 2015

We established nude­ mouse models of human pancreatic cancer with surgical orthotopic implantation of the human BxPC­3 pancreatic cancer for fluorescence-guided surgery (FGS). After 2 weeks, mice then underwent bright-­light surgery (BLS) or FGS 24 hours after intravenous injection of anti CEA­-Alexa Fluor 488. Completeness of resection was assessed from postoperative imaging. Mice were followed postoperatively until premorbid to determine disease-free survival (DFS) and overall survival (OS). Complete resection was achieved in 92% of mice in the FGS group compared to 45.5% in the BLS group (p = 0.001). FGS resulted in a smaller postoperative tumor burden (p = 0.01). Cure rates with FGS compared to BLS improved from 4.5 to 40%, respectively (p = 0.01), and 1­year postoperative survival rates increased from 0% with BLS to 28% with FGS (p = 0.01). Median DFS increased from 5 weeks with BLS to 11 weeks with FGS (p = 0.0003). Median OS increased from 13.5 weeks with BLS to 22 weeks with FGS (p = 0.001). FGS resulted in greater cure rates and longer DFS and OS using a fluorophore-­conjugated anti ­CEA antibody. The present results demonstrate that FGS has potential to improve the surgical treatment of pancreatic cancer.

Citation Format: Cristina A. Metildi, Sharmeela Kaushal, George A. Luiken, Robert M. Hoffman, Michael Bouvet. Pancreatic cancer fluorescence-­guided surgery with a fluorophore­-conjugated antibody to carcinoembryonic antigen (CEA) improves surgical resection and increases disease-free and overall survival in orthotopic mouse models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 216. doi:10.1158/1538-7445.AM2015-216
Abstract 2162: Loss of ATF3 promotes akt activation and prostate cancer development in a pten knockout mouse model
Authors: Wang Ziyan , Xu Dong , Yan Chunhong
Published: August 2015

Activating transcription factor 3 (ATF3) responds to diverse cellular stresses, and regulates oncogenic activities (e.g., proliferation, survival and migration) through direct transcriptional regulation or protein-protein interactions. Although aberrant ATF3 expression was often found in human cancers, the role of ATF3 in tumorigenesis is poorly understood. Here we demonstrate that ATF3 suppresses the development of prostate cancer induced by knockout of the tumor suppressor Pten in mouse prostates. Whereas oncogenic stress caused by Pten loss induced ATF3 expression in prostate epithelium, we found that ATF3 deficiency increased cell proliferation but inhibited apoptosis, leading to early onset of mouse prostatic intraepithelial neoplasia and the progression of prostate lesions to invasive adenocarcinoma. Importantly, loss of ATF3 appeared to promote activation of the oncogenic AKT signaling evidenced by high levels of phosphorylated AKT and S6 proteins in ATF3-null prostate lesions. In line with these in vivo results, knockdown of ATF3 expression in human prostate cancer cells by sgRNA-mediated targeting activated AKT, elevated NF-KB nuclear translocation and increased expression of a broad of matrix metalloproteinase such as MMP-9, MMP-10. By zymography and q-PCR assays, we found that MMP-9 expression increased in ATF3-knockdown DU145 and PC3 cells. Also from luciferase reporter result, we further confirmed that loss of ATF3 could elevate MMP-9 expression via activating NF-KB. And in vivo immunochemistry results also show increased MMP9 expression and discontinuous a-SMA staining along prostate glands. These results were well anticipated that ATF3 deficiency leads to activated AKT signaling, which further activates IKK-A to increase nuclear translocation of NF-KB, which in turn binds to the MMP-9 promoter and induces MMP-9 expression in ATF3-downregulated prostate cancer cells, then promotes invasion in Pten-lost mouse prostates. Our results thus link ATF3 to the AKT signaling, and provide the first genetic evidence supporting that ATF3 is a tumor suppressor for a subset of prostate cancers harboring dysfunctional Pten.

Citation Format: Ziyan Wang, Dong Xu, Chunhong Yan. Loss of ATF3 promotes akt activation and prostate cancer development in a pten knockout mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2162. doi:10.1158/1538-7445.AM2015-2162
Abstract 2174: Identification of genetic factors contributing to development of common cancers through tissue-specific protein interaction analysis
Authors: Qian David C. , Byun Jinyoung , Han Younghun , Hunter David J. , Henderson Brian E. , Eeles Rosalind , Haiman Christopher A. , Easton Douglas F. , Hung Rayjean J. , Amos Christopher I.
Published: August 2015

In the genetic study of diseases, pathway based analysis captures more of the variability in risk than single-variant analysis, and also provides a meaningful framework for organizing and interpreting findings. Here, we augment the biologic insights provided by pathway based analysis of GWAS results by incorporating tissue-specific protein interactions. From large GWAS meta-analyses of lung cancer (12,160 cases/16,838 controls), breast cancer (15,748 cases/18,084 controls), and prostate cancer (14,160 cases/12,724 controls), we determined the tissue-specific interactomes of proteins expressed from genes containing independently associated variants. The pathways with statistical overrepresentation of proteins in each network were evaluated across the three cancers. Our results show that pathways implicated in the development of all three cancers or two out of the three cancers tend to be broad, essential cellular processes required for growth and survival. The most significant examples include the nerve growth factor (P = 1.65 × 10^-47), epidermal growth factor (P = 1.22 × 10^-37), and stem cell factor/Kit (P = 4.47 × 10^-35) signaling cascades. However, within these shared pathways, the proteins encoded by genes with risk-conferring variants generally differ from cancer to cancer. Pathways found to be unique for a single cancer focus on more specific cellular functions, such as leptin signaling in breast cancer (P = 1.24 × 10^-6) and platelet sensitization by low-density lipoprotein in prostate cancer (P = 3.80 × 10^-6). Taken together, we demonstrate a new method of pathway based analysis following GWAS that considers tissue-specific protein interactions. For cancers of the lung, breast, and prostate, we validate known susceptibility pathways and identify previously unexplored ones, as well as characterize each cancer by its unique pathways.

Citation Format: David C. Qian, Jinyoung Byun, Younghun Han, David J. Hunter, Brian E. Henderson, Rosalind Eeles, Christopher A. Haiman, Douglas F. Easton, Rayjean J. Hung, Christopher I. Amos. Identification of genetic factors contributing to development of common cancers through tissue-specific protein interaction analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2174. doi:10.1158/1538-7445.AM2015-2174
Abstract 220: New computerized image analyzing technique for analysis of bone marrow microvessel density in multiple myeloma
Authors: Sun-Young Kong , Lee Nuri , Kim Young Jae , Sohn Ji Yeon , Lee Hyewon , Eom Hyeon-Seok , Kim Kwang Gi
Published: August 2015

Background: Angiogenesis is important in proliferation and survival of neoplastic cells in multiple myeloma (MM). Microvessel density (MVD) is a useful angiogenesis marker, which can be detected by anti-CD34 immunohistochemical stain. However, counting the MVD of bone marrow (BM) by light microscopy has a limitation in standardization due to inter-observer differences. Consistent method for MVD assessment has been required to resolve the variation in number. Here we developed the MVD counting method using computerized image analyzer (CIA) and light microscopy.

Methods: A total of 21 patients with primary diagnosed with MM were evaluated. MVD was assessed by screening three hot spots of CD34 positive vessels on BM biopsy section (x40 magnification). MVD was measured by three different ways. First, the number of vessels was counted using light microscope at the time of diagnosis by several hematopathologists (MVD-A). Second, the total count of microvessels per 14435.2 μm2 (1388 × 1040μm) of marrow area was measured by a Zeiss microscope using AxioVison LE Image Analyzer with developed image analyzer program (MVD-B). In addition, manual count of captured image by CIA with one observer was examined (MVD-C). Software was developed using Microsoft visual studio (ver. 2010, Microsoft, USA) and VTK (ver. 5.10.0, Kitware, USA) library. Captured image was labeled via pre-processing for image enhancement and seed region growing algorithm to detect the MVD candidates. Using histogram texture analysis, each candidate was determined to MVD.

Results: The range of MVD was as followings: MVD-A (range 3-52; mean 16.5; SD 12.8), MVD-B (range 4-38; mean 19.6; SD 10.0), and MVD-C (range 5-34; mean 14.8; SD 7.8).There was a significant correlation between theses angiogenesis parameters. Pearson's correlation coefficient of MVD-A with MVD-B was 0.535 (P = 0.01), MVD-A with MVD-C was 0.581 (P < 0.01), and MVD-B with MVD-C was 0.897 (P < 0.01).

Conclusions: We developed the computer aided diagnostic tools to measure MVD more objectively. MVD count using image analyzer was well correlated with manual count and more accurate than MVD count on light microscope. Further studies with continuous upgrades on counting program are now in progress. Finally, we tried to apply this method in practice to predict the prognostic impact on MM patient at the time of diagnosis.

Citation Format: Kong Sun-Young, Nuri Lee, Young Jae Kim, Ji Yeon Sohn, Hyewon Lee, Hyeon-Seok Eom, Kwang Gi Kim. New computerized image analyzing technique for analysis of bone marrow microvessel density in multiple myeloma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 220. doi:10.1158/1538-7445.AM2015-220
Abstract 2216: A novel G-quadruplex formed in the proximal P1 promoter of bcl-2 gene is a gene suppressor
Authors: Onel Buket , Carver Megan , Yang Danzhou
Published: August 2015

Deregulation of bcl-2, an anti-apoptotic protein, is associated with several human diseases such as tumorigenesis, neurodegeneration, and autoimmune deficiencies. DNA G-quadruplex secondary structures that form in the G-rich proximal promoter regions have been found to play an important role in gene transcriptional regulation. We discovered a novel GC-rich sequence in the immediate proximal P1 promoter of the bcl-2 gene (bcl2P1Pu29), which forms a thermodynamically stable, intramolecular G-quadruplex (bcl2P1G4) that represses bcl-2 gene transcription. Dimethyl sulfate footprinting studies indicated that this bcl2 P1 promoter G-rich region forms G-quadruplexes under physiologically relevant ionic conditions. This was shown by clear DMS protection of guanine N7, which is characteristic of G-quadruplex formation. NMR spectroscopic study of bcl2P1Pu29 showed the formation of a well-defined major G-quadruplex structure under physiologically relevant ionic conditions, as evident by sharp peaks in the imino proton region. Interestingly, NMR data also indicated the presence of Watson-Crick base-pairing, suggesting a potential unique loop conformation. Circular dichroism (CD) studies suggested that bcl2P1G4 adopts parallel-stranded folding. Both NMR and CD data indicated a monomeric structure of bcl2P1G4 with high thermo-stability. Using a luciferase assay, we have shown that TMPyP4, a planar cationic G-quadruplex-interactive compound, significantly inhibits the promoter activity through this G-rich bcl-2 P1 promoter region, while TMPyP2, a positional isomer of TMPyP4 which is a poor G-quadruplex-interactive compound, did not show a significant effect. Our results suggest that this novel intramolecular G-quadruplex formed in the bcl-2 proximal promoter region appears to be a suppressor of gene transcription, which could be a potential molecular target for small molecules to modulate bcl-2 gene transcription.

Citation Format: Buket Onel, Megan Carver, Danzhou Yang. A novel G-quadruplex formed in the proximal P1 promoter of bcl-2 gene is a gene suppressor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2216. doi:10.1158/1538-7445.AM2015-2216
Abstract 2225: c-Myc and Frizzled 8 play a major role in the regulation of cancer stem cells and drug resistance in triple-negative breast cancer
Authors: Yin Shuping , Cheriyan Vino T. , Rishi Arun K. , Reddy Kaladhar B.
Published: August 2015

Triple-negative breast cancer (TNBC) studies have shown that neoadjuvant chemotherapy before surgery was effective in the minority of women, whereas the majority that had a residual tumor had a relatively poor outcome. We have previously shown that cisplatin plus TRAIL treatment was most effective compared to PARP -inhibitors, pacliotaxol, dacelotaxol or cisplatin alone in TNBC cells. To identify the mechanism by which residual cancer cells survive chemotherapy, we initially performed gene expression profiling using the CRL2335 and MDA-MB-468 TNBC cells before and after treatment with cisplatin plus TRAIL. Gene expression prolife revealed that cisplatin plus TRAIL significantly inhibited major signaling pathways related with cancer stem cells (CSCs) such as Notch, Hedgehog, TGFβ and most of the Wnt/β-catenin signaling when compared with untreated control group. However, residual cells after cisplatin plus TRAIL treatment showed a significant increase in the expression of Frizzled 8 (FZD8 -one of the Wnt receptors) and c-Myc. In vivo, animal studies indicate that cisplatin plus TRAIL treatments predominantly eliminated non-cancer stem cells (non-CSCs), as demonstrated by whole-body fluorescent imaging of mice injected with mammosphere-forming CRL2335 cells that were stably transfected with dsRed. This led to CSCs enrichment in residual tumors, as confirmed by immunostaining for CSC markers. Increase in FZD8 and c-Myc expression was also observed in residual tumors following treatment with cisplatin and TRAIL. In this study, we have found that c-Myc overexpression increased FZD8 expression, and its downstream signaling and together protect the cells from drug mediated cell death compared to vector transfected cells. c-Myc over-expressing cells do not show increased mammosphere formation; the numbers of CSCs per mammosphere are much higher compared to vector transfected controls. CDK inhibitor (dinaciclib) enhanced the cell death in c-Myc over expressing non-CSCs with minimal effect on CSCs. Inhibition of c-Myc using siRNA reduced c-Myc, FZD-8 and CSCs and enhanced sensitivity of cells to cisplatin plus TRAIL. Taken together, our findings suggest that c-Myc may play a major role in mediating CSC survival and resistance to chemotherapy, making it a potential target to enhance chemotherapeutic efficacy in patients with TNBC.

Citation Format: Shuping Yin, Vino T. Cheriyan, Arun K. Rishi, Kaladhar B. Reddy. c-Myc and Frizzled 8 play a major role in the regulation of cancer stem cells and drug resistance in triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2225. doi:10.1158/1538-7445.AM2015-2225
Abstract 2261: DLGAP5, MAT1A, SKA3, and ZMYM5 are novel susceptibility genes for aggressive prostate cancer
Authors: Lee Minnkyong , Williams Kendra A. , Hu Ying , Andreas Jonathan , Patel Shashankkumar J. , Zhang Suiyuan , Crawford Nigel PS
Published: August 2015

Prostate cancer is a common disease with approximately 233,000 men estimated to be diagnosed in the United States alone in 2014. Yet, it is usually an indolent disease with only 13% of patients succumbing to prostate cancer, and the molecular determinants of aggressive prostate cancer remain unclear. Previously, we reported that germline variation influences disease aggressiveness in the C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model. These mice develop neuroendocrine prostate tumors similar to a subset of human prostate tumors associated with poor outcomes. Here, we used a multifaceted approach to identify candidate genes for susceptibility to aggressive prostate tumorigenesis and metastasis using the TRAMP mouse model. Candidate prostate cancer metastasis susceptibility genes were identified through quantitative trait locus (QTL) mapping in 201 (TRAMP x PWK/PhJ) F2 males. Two aggressive disease QTLs were identified; one for lymph node metastasis burden on chromosome 12 (LOD = 5.86) and one for distant metastasis-free survival on chromosome 14 (LOD = 4.41). Correlation analysis using microarray data derived from 27 (TRAMP x PWK/PhJ) F2 prostate tumors identified 35 metastasis-associated transcripts within the two loci. The role of these genes in susceptibility to aggressive human prostate cancer was analyzed in two different datasets. First, logistic regression and survival analyses in human prostate cancer gene expression datasets demonstrated that the expression levels of 5 of the 35 candidate genes was associated with both an increased risk of aggressive disease and a poorer disease-free survival. Second, four of these genes - DLGAP5, MAT1A, SKA3, and ZMYM5 - harbored SNPs associated with aggressive tumorigenesis in the PLCO/CGEMS GWAS cohort of 1,172 prostate cancer patients. This approach, novel to the prostate cancer field, demonstrates how mouse models can be used to identify aggressive disease susceptibility genes, and gives new insight into the molecular mechanisms of aggressive disease.

Citation Format: Minnkyong Lee, Kendra A. Williams, Ying Hu, Jonathan Andreas, Shashankkumar J. Patel, Suiyuan Zhang, Nigel PS Crawford. DLGAP5, MAT1A, SKA3, and ZMYM5 are novel susceptibility genes for aggressive prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2261. doi:10.1158/1538-7445.AM2015-2261
Abstract 2321: A hematopoietic stem cell factor drives brain tumor initiating cell genesis through Notch signaling
Authors: Kim Sunghak , Kim Eun-Jung , Cheol Gyu Park , Oh Se-Yeong , Mutsuko Minata , Kim Hyunggee , Nakano Ichiro
Published: August 2015

Glioblastomas (GBMs) maintain their cellular heterogeneity with glioma stem cells (GSCs) producing a variety of tumor cell types. Here, we interrogated the oncogenic roles of Lim domain only 2 (LMO2) in GBM and GSCs in mice and human. High expression of LMO2 was found in human patient-derived GSCs compared to the differentiated progeny cells. LMO2 is required for GSC growth both in vitro and in vivo, as shRNA-mediated LMO2 silencing attenuated tumor growth derived from human GSCs. Further, LMO2 is sufficient to induce stem cell characteristics (stemness) in mouse pre-malignant astrocytes, as forced LMO2 expression facilitated in vitro and in vivo growth of astrocytes derived from Ink4a/Arf null mice and acquisition of GSC phenotypes. A subset of mouse and human GSCs converted into vascular endothelial-like tumor cells both in vitro and in vivo, which phenotype was attenuated by LMO2 silencing and promoted by LMO2 overexpression. Mechanistically, the action of LMO2 for induction of glioma stemness is mediated by transcriptional regulation of Jagged1 resulting in activation of the Notch pathway, whereas LMO2 directly occupies the promoter regions of the VE-cadherin gene for a gain of endothelial cellular phenotype. Subsequently, selective ablation of human GSC-derived VE-cadherin-expressing cells attenuated vascular formation in mouse intracranial tumors, thereby significantly prolonging mouse survival. Clinically, LMO2 expression was elevated in GBM tissues and inversely correlated with prognosis of GBM patients. Taken together, our findings describe novel dual roles of LMO2 to induce tumorigenesis and angiogenesis, and provide potential therapeutic targets in MGs.

Citation Format: Sunghak Kim, Eun-Jung Kim, Park Cheol Gyu, Se-Yeong Oh, Minata Mutsuko, Hyunggee Kim, Ichiro Nakano. A hematopoietic stem cell factor drives brain tumor initiating cell genesis through Notch signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2321. doi:10.1158/1538-7445.AM2015-2321
Abstract 2322: The Hippo signaling pathway mediates BMP inhibition of cancer stem cells
Authors: Chartier Cecile , Ma Shirley , Antic Dragana , Quintana Elsa , Raval Janak , Wang Min , Dee-Hoskins Cristina , Kapoun Ann , Hoey Tim , Lewicki John , Gurney Austin
Published: August 2015

Multiple developmental signaling pathways cooperatively regulate cancer stem cell (CSC) function. Among them, the BMP pathway has been implicated in brain CSC tumorigenicity. To examine whether BMP is a general regulator of CSCs across diverse organ systems we utilized lentivirus-mediated BMP4 expression in patient-derived xenograft models in NOD/SCID mice. The prevention of tumor growth was observed in breast, colon, ovarian, and melanoma models. Similarly, adenovirus-mediated systemic delivery of BMP4 inhibited tumor growth, dramatically affected CSC-specific cell surface markers and decreased CSC frequency. Microarray analysis of BMP4-treated tumors showed that a set of genes encoding for components of the actin cytoskeleton was significantly enriched amongst regulated genes and identified Hippo pathway genes as potential mediators of the BMP4 anti-tumor response with up-regulation of SAV1 and LATS2 and down-regulation of BIRC5. Hippo signaling regulates organ size, stem cell biology and cancer through a protein kinase complex including MST1/2, SAV1, and LATS1/2, which phosphorylates the downstream nuclear effector YAP/TAZ. Phosphorylated YAP/TAZ is unable to induce the expression of cell proliferation and anti-apoptotic genes through interaction with transcriptional co-activators such as TEAD. The Hippo pathway, known to cross-talk with BMP signaling at multiple levels, recently emerged as a cellular sensor of the physical environment through the actin cytoskeleton. We thus asked whether modulating Hippo signaling affected tumor growth. YAP1 overexpression resulted in tumor growth decrease. A deletion variant of YAP, YAP1-291, possessing a deletion of the transactivation domain has been shown to act as a dominant-negative through binding to TEAD. Intriguingly YAP1-291 was also sufficient to strongly attenuate growth of diverse BMP4-sensitive and BMP4-insensitive tumor models suggesting that transactivation through TEAD is not the only route by which YAP modulates tumor growth. YAP anti-proliferative properties were reported before. In order to reconcile the dual function of YAP in cancer, we propose that the transcriptional co-activator's TEAD-mediated oncogenic function may be counter-balanced by its potentiation of SMAD signaling in response to BMP.

Citation Format: Cecile Chartier, Shirley Ma, Dragana Antic, Elsa Quintana, Janak Raval, Min Wang, Cristina Dee-Hoskins, Ann Kapoun, Tim Hoey, John Lewicki, Austin Gurney. The Hippo signaling pathway mediates BMP inhibition of cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2322. doi:10.1158/1538-7445.AM2015-2322
Abstract 2328: Targeting self-renewal in human colorectal cancer by inhibiting SUMOylation
Authors: Du Li , Chen Yuan
Published: August 2015

Colorectal cancer is the third most commonly diagnosed cancer and the second leading cause of cancer death in the US. It is well recognized that cancer stem cells (CSCs) are responsible for treatment resistance, tumor metastasis and relapse, which cause treatment failure. SUMOylation is known to be important for pluripotency and is dysregulated in cancers to drive tumor growth and metastasis. Therefore, we investigate the role of SUMOylation in colorectal cancer CSC maintenance and self-renewal.

In this study, we established that aldehyde dehydrogenase 1 (ALDH1) activity is a valid marker of colorectal CSCs, because ALDH+ cells in HT29 displayed CSC properties including self-renewal, clonal formation and tumor initiation in NSG xenograft mouse model, while ALDH- cells did not. Profiling ALDH+ and ALDH- cells showed that SUMO-activating enzyme (SAE) is highly enriched in ALDH+ cells along with enhanced global SUMOylation. Overexpression of the catalytic subunit of SAE, SAE2, increased the population of ALDH+ cells while knockdown of SAE2 decreased the population of ALDH+ cells. This result suggests that increased SAE level promotes stemness in colorectal cells. To characterize CSC maintenance and self-renewal quantitatively, we performed limited dilution assays in vitro with sphere initiation and in vivo using mouse xenograft models. In in vitro assays, we observed 4-fold reduction in CSC self-renewal frequency in SAE2 knockdown cells compared to control cells in the colorectal cancer cell line HT29. In four colorectal cancer patient-derived primary culture cells, the frequency was 2∼6 fold lower in SAE2 knockdown groups than control. In the in vivo experiment, SAE2 knockdown group showed 12-fold reduction in self-renewal frequency of HT29 cells. These data demonstrate that silencing of SAE2 causes a significant decrease in stemness of colorectal cancer cells, both in HT29 cell line and primary human tumors. Mechanistic analysis regarding how SUMOylation regulate colorectal cancer CSC is underway and will be discussed.

In conclusion, our study indicates that SUMOylation, in particular the SAE, is an important regulator of colorectal CSC maintenance and self-renewal. Therefore, the data suggests that targeting SAE is a potential strategy to inhibit colorectal CSC.

Citation Format: Li Du, Yuan Chen. Targeting self-renewal in human colorectal cancer by inhibiting SUMOylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2328. doi:10.1158/1538-7445.AM2015-2328
Abstract 236: Autocrine regulation of stress-induced cytotoxic Alu RNA expression in TNBC cells
Authors: Singhal Ashutosh , Mishra Smita , Chaudhuri Gautum
Published: August 2015

Breast tumor cells are often subject to various forms of proteotoxic stress in their microenvironment mainly presented by the stromal cells. We found that the highly aggressive breast cancer cells of the triple negative subtype (TNBC) are significantly more tolerant to environmental stress than the non-aggressive breast cancer cells. Major objective of the research presented here is to understand the mechanism of induction of cytotoxic Alu RNA in the stressed breast cancer cells and how the TNBC cells pacify this non-coding RNA-mediated cytotoxicity. We present evidence that stress-induced activation of HSF1 in the breast cancer cells stimulate the RNA polymerase III-mediated transcription of Alu RNA genes in these cells resulting in induction of severe cytotoxicity and apoptosis of these cells. Highly aggressive stress-tolerant TNBC cells secrete the matrix metalloprotease MMP1 which activates the G-protein coupled receptor PAR1 on their cell surface. Activation of PAR1 through the Galpha-q/PIPLC/PIP3 pathway raises the cytosolic concentration of Ca2+ thereby activating the Ca2+-dependent protease mu-calpain. Mu-calpain then proteolytically degrades the RNA polymerase III transcription factor TFIIIC110 at the PES sequence. As TFIIIC110 is required for the transcription of Alu RNA genes, its degradation ameliorates stress-induced elevation of Alu RNA and thus its cytotoxicity. We propose that inhibition of MMP1, PAR1 or mu-calpain in the TNBC cells will prevent this pacification mechanism promoting stress-induced demise of these cells. This research is supported in part by NIH/NCI grant 1R21CA181920-01 to GC.

Citation Format: Ashutosh Singhal, Smita Mishra, Gautum Chaudhuri. Autocrine regulation of stress-induced cytotoxic Alu RNA expression in TNBC cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 236. doi:10.1158/1538-7445.AM2015-236
Abstract 2436: Understanding the molecular mechanism of targeted kinase inhibitor resistance mediated by the FGFR1 gatekeeper mutation
Authors: Sohl Christal D. , Ryan Molly , Luo BeiBei , Frey Kathleen , Anderson Karen S.
Published: August 2015

Human fibroblast growth factor receptors (FGFRs) can serve as drivers of oncogenesis and are a target of several inhibitors in clinical trials. Unfortunately, resistance severely hampers the long-term success of tyrosine kinase inhibitors (TKIs), with mutations at the gatekeeper residue often serving as an early means of resistance to cancer therapy, resulting in tumor progression. Here we show the first crystal structures of the FGFR1 gatekeeper mutation, V561M FGFR1, and use kinetic and structural methods to explore the mechanisms of the catalytic activation we observe for this mutation. We show that while the FGFR1 gatekeeper mutation confers resistance to two inhibitors currently in clinical trials, the extent of change in efficacy differ, indicating two unique resistance profiles. We show that some affinity for one inhibitor is maintained by the V561M mutation due to a flexible linker that allows multiple binding conformations. This is the first example showing the same inhibitor binding in unique ways to the WT and gatekeeper mutant forms of FGFR, highlighting some regions of drug design flexibility both within the binding pocket and in the linker of the inhibitor. Identifying this flexibility in the context of the structural features of an active conformation of V561M FGFR1, the form most kinase inhibitors target, will provide critical insights for designing future inhibitors effective against the FGFR gatekeeper mutations.

Citation Format: Christal D. Sohl, Molly Ryan, BeiBei Luo, Kathleen Frey, Karen S. Anderson. Understanding the molecular mechanism of targeted kinase inhibitor resistance mediated by the FGFR1 gatekeeper mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2436. doi:10.1158/1538-7445.AM2015-2436
Abstract 2455: Differences in dynamic modulation of CoQ10 redox status and intracellular localization between non-disease and cancer cell lines
Authors: Krumova Katerina , Garg Sumit , Dhavala Sirisha , Vishnudas Vivek K. , Kiebish Michael A. , Sarangarajan Rangaprasad , Narain Niven R.
Published: August 2015

Although it is well recognized that Coenzyme Q10 (CoQ10) is present in all cellular membrane structures, its effects on cellular functions other than mitochondrial regulation is poorly understood. The present study was aimed at gleaning insights into effect on CoQ10 on sub-cellular metabolism, regulation of the redox status, as well as gene regulation. A series of fluorescent probes were developed with the ability to report on the consequence of specific biological events in which CoQ10 was involved. The probes constituted fluorescently labeled CoQ10 analogues designed to track the distribution, biochemical and redox behaviour of CoQ10 in a non-invasive manner providing real time information in live cells. Compartmentalization of fluorescent labeled CoQ10 probe incorporated within BPM 31510 liposomal formulation was characterized in human derived primary non-disease and cancer cell lines. Cellular distribution of the fluorescent CoQ10 varied depending upon cell type. The fluorescent CoQ10 probes were designed and utility to provide a time resolved assessment on the redox state of CoQ10 in whole cells and organelles as a measure of the cell's overall and intracellular location specific metabolic micro-environments. The ratio of reduced and oxidized form of fluorescent CoQ10 was also examined in relation to ROS production in cancer cells. The overall results describes the presence of a constant redox flux influencing the redox state of CoQ10 within cancer and normal cells with significant variation depending on cell type and disease status.

Citation Format: Katerina Krumova, Sumit Garg, Sirisha Dhavala, Vivek K. Vishnudas, Michael A. Kiebish, Rangaprasad Sarangarajan, Niven R. Narain. Differences in dynamic modulation of CoQ10 redox status and intracellular localization between non-disease and cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2455. doi:10.1158/1538-7445.AM2015-2455
Abstract 2478: A mAb with in vivo therapeutic activity against spontaneous breast tumors in FVB-neuN mice recognizes SLP-2
Authors: Wang Li-Xin , Berk Michael , Plautz Gregory E.
Published: August 2015

BCSC (breast cancer stem cells) derived from FVB-neuN mice bearing primary breast tumors were used to vaccinate mice and vaccine-draining lymph node T cells were adoptively transferred to mice with advanced primary tumors. Treated mice experienced tumor regression and importantly a concomitant serologic response was induced to an antigen that is expressed by BCSC even in Neu-antigen loss tumor cells. We generated a specific mAb (mAb2) reactive against a membrane antigen on BCSC tumorsphere cultures derived from NeuN spontaneous breast tumors but not the counterpart adherent cultures. In vivo administration of mAb2 inhibited progression of macroscopic primary tumors in Her-2/neu transgenic mice. We further observed that the molecule bound by mAb2 is expressed on proliferating but not growth-arrested C2D1-tumorspheres and this antigen is re-expressed when cells are digested into single cell suspension and re-fed with fresh medium. Similar results are observed with 4T1-tumorsphere cells, an additional breast cancer cell line arising initially in BALB/c mice. Interestingly, the target of mAb2 is also down-regulated following irradiation-induced growth arrest of tumor cells. To identify the antigen recognized by mAb2, cultured C2D1-tumorsphere or 4T1-tumorspheres were cells were lysed and the supernatant was immunoprecipitated with mAb2. The proteins captured by mAb2 were separated by 10% SDS-PAGE and visualized by silver staining. The bands of interest were excised then subjected to liquid chromatography-mass spectrometric (LC-MS/MS) analysis. A ∼40 KDa protein was detected in all five experiments from either C2D1-tumorsphere or 4T1-tumorsphere cells. Stomatin-like protein 2 (SLP-2) a 38.5 KDa protein was consistently identified whereas 14 other proteins were sporadically found. Western blots of mAb2 immunoprecipitates from C2D1 or 4T1-tumorsphere lysates were probed with two distinct commercial antibodies against SLP-2 and we confirmed its presence. To validate SLP-2 as the target, an expression vector was transfected into NIH 3T3 cells and FACS analysis demonstrated 19% positive cells versus 3.6% for un-transfected 3T3 cells. SLP-2 overexpression is associated with tumor metastasis and poor prognosis in several types of human tumors. Our results indicate that the antigen recognized by this auto-antibody might have immunodiagnostic or immunotherapeutic potential.

Citation Format: Li-Xin Wang, Michael Berk, Gregory E. Plautz. A mAb with in vivo therapeutic activity against spontaneous breast tumors in FVB-neuN mice recognizes SLP-2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2478. doi:10.1158/1538-7445.AM2015-2478
Abstract 2509: Human anti-MUC1 antibodies elicited by a prophylactic cancer vaccine for mAb and CAR-modified T cell immunotherapies
Authors: Lohmueller Jason J. , Sato Shuji , Cheung Wan Cheung , Chu Isabel , Popova Lana , Manning Christopher A. , Crosby Katherine , Grange Christopher , Ham James D. , Polakiewicz Roberto D. , Finn Olivera J.
Published: August 2015

MUC1 is a tumor-associated protein that is abnormally expressed on human adenocarcinomas. In our recent clinical trial, individuals at-risk for colonic adenocarcinoma received a prophylactic MUC1 cancer vaccine. Many individuals responded to the vaccine by producing high titers of anti-MUC1 IgG antibodies (Kimura, 2013). We isolated a panel of these antibodies and are seeking to develop them as antibody and chimeric antigen receptor (CAR) T cell cancer immunotherapies. To this end we used a novel method combining proteomics and next-gen DNA sequencing to identify and recombinantly produce 12 high-affinity anti-MUC1 antibodies from the sera of vaccinated individuals. By ELISA we determined that the antibodies bind with high-affinity to diverse epitopes within the MUC1 vaccine peptide. We also found that several of the antibodies can stain MUC1+ pancreas, colon, and breast cancer cell lines, but not a cell line expressing normal MUC1. We then determined that the antibodies could stain MUC1+ human pancreas and colon tumor samples, but not normal tissues. Having established tumor-specificity we have begun assessing the antibodies in a series of functional assays. In preliminary in vitro testing we determined that several of the antibodies can be internalized by MUC1+ cells making them potential candidates for toxin delivery as antibody drug conjugates (ADCs). Additionally, we found that two of the antibodies are capable of efficiently performing complement-dependent cytotoxicity (CDC) on MUC1+ cells. Finally, we have constructed CAR lentiviral expression constructs using the variable regions of several of the antibodies as targeting domains and are testing their ability to mediate MUC1-specific T cell activation, cytokine production, and target cell lysis. As the antibodies are human-derived, indication of efficacy in pre-clinical testing has the potential to directly enable clinical trials and hopefully approval of these reagents for therapy of cancer patients.

Citation Format: Jason J. Lohmueller, Shuji Sato, Wan Cheung Cheung, Isabel Chu, Lana Popova, Christopher A. Manning, Katherine Crosby, Christopher Grange, James D. Ham, Roberto D. Polakiewicz, Olivera J. Finn. Human anti-MUC1 antibodies elicited by a prophylactic cancer vaccine for mAb and CAR-modified T cell immunotherapies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2509. doi:10.1158/1538-7445.AM2015-2509
Abstract 2542: Lurbinectedin (PM01183) synergizes in vivo the antitumor activity of doxorubicin in SCLC tumor xenografts
Authors: Guillen Maria Jose , Cataluña Oscar , Palomares Mandy , Lopez Raquel , Nuñez Praxedes , Cuevas Carmen , Aviles Pablo M.
Published: August 2015

Background: Lurbinectedin (PM01183) is a synthetic tetrahydroisoquinoline alkaloid currently evaluated as single agent and in combination in phase I and II clinical trials for solid tumors and hematological malignancies. In living cells, PM01183-DNA adducts stall replication and transcription giving rise to double strand breaks, inducing accumulation of cells in the S-phase of the cell cycle and triggering apoptosis. Studies aimed at defining the in vivo synergism of PM01183 and doxorubicin in the treatment of Small Cell Lung Cancer (SCLC) xenografted tumors are presented here.

Material and Methods: Athymic nude mice were implanted with SCLC cells (NCI-H82 or NCI-H526). Mice bearing tumors (ca. 150 mm3) were allocated (N = 6-8/group) to experimental groups, namely: placebo; PM01183 at MTD (0.18 mg/kg), 0.75MTD, 0.5MTD and 0.25MTD; doxorubicin at their MTD (8.0 mg/kg), 0.75MTD, 0.5MTD and 0.25MTD; and, PM01183 combined with doxorubicin at (1+1), (0.75+0.75), (0.50+0.50) and (0.25+0.25) MTDs. All treatments were given intravenously once per week during the placebo-treated survival time. The antitumor effect induced by the treatments was analyzed using two-tailed Mann-Whitney U test. Also, the synergism was defined by the combination index (CI) determined by the CI-isobol method.

Results: In both experiments, the combination of PM01183 and doxorubicin resulted in stronger antitumor effect (P<0.05) than that obtained with the more active single agent at the highest dose (MTD level). Analyses of the results by the CI-isobol method indicated that, after the treatment with PM01183 plus doxorubicin in the SCLC models resulted in an additive (at Fa = 0.97, CI = 1.05) or synergistic (at Fa = 0.97, CI = 0.66) effect.

Conclusion: An improved (additive or synergism) in vivo antitumor activity was recorded after the treatment with PM01183 plus doxorubicin of mice bearing SCLC xenografted tumors.

Citation Format: Maria Jose Guillen, Oscar Cataluña, Mandy Palomares, Raquel Lopez, Praxedes Nuñez, Carmen Cuevas, Pablo M. Aviles. Lurbinectedin (PM01183) synergizes in vivo the antitumor activity of doxorubicin in SCLC tumor xenografts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2542. doi:10.1158/1538-7445.AM2015-2542