bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4853: NRP2b, a unique isoform of NRP2, promotes aggressive lung cancer phenotypes
Authors: Dimou Anastasios , Nasarre Patrick , Nair-Menon Joyce , Cappuzzo Federico , Landi Lorenza , D'Incecco Armida , Uramato Hidetaka , Yoshida Takeshi , Haura Eric , Gooz Monica , Armeson Kent , Gemmill Robert , Drabkin Harry
Published: July 2017

Neuropilins (NRPs) 1 and 2 are highly-related receptors for class 3 semaphorins, and interact with heparin-binding growth factors and their receptors (e.g. HGF-MET, VEGF-VEGFR2 etc.). We previously reported that NRP2 is upregulated by TGFβ and is required for its pro-tumorigenic activity. Here, we show that this upregulation preferentially involves NRP2b, a largely uninvestigated isoform encoding a divergent, yet highly-conserved C-terminus. Importantly, using a panel of lung cancer cell lines and orthotopic metastasis model, NRP2b promoted migration, invasion, metastasis and tumorsphere formation, whereas the prototype receptor, NRP2a had opposite effects. TGFβ-mediated resistance to gefitinib in EGFR mutated tumors also was dependent on NRP2b expression. In addition, NRP2b, but not NRP2a, linked MET activation by HGF to AKT phosphorylation. Mechanistically, in co-immunoprecipitation assays, we found that NRP2a robustly interacted with PTEN, while the interaction with NRP2b was weak. At the clinical level, NRP2b expression was commonly upregulated in patient lung cancer samples and this upregulation was a significant poor-prognostic factor. Collectively, these data indicate that NRP2b plays an important role in lung cancer invasion, metastasis and EGFR inhibitor resistance. Furthermore, isoform-specific interactions between NRP2 and PTEN may be responsible for the observed differences in MET signaling in response to HGF.

Citation Format: Anastasios Dimou, Patrick Nasarre, Joyce Nair-Menon, Federico Cappuzzo, Lorenza Landi, Armida D'Incecco, Hidetaka Uramato, Takeshi Yoshida, Eric Haura, Monica Gooz, Kent Armeson, Robert Gemmill, Harry Drabkin. NRP2b, a unique isoform of NRP2, promotes aggressive lung cancer phenotypes [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 4853. doi:10.1158/1538-7445.AM2017-4853
Abstract 4858: AK4 promotes colorectal cancer progression and metastasis
Authors: Yu Yau Hei , Tong Joanna Hung Man , Pan Yi , Lung Raymond Wai Ming , To Ka Fai , Chan Anthony Wing Hung
Published: July 2017

Metastatic colorectal cancer (CRC) is a fatal disease with a poor prognosis. It is crucial to understanding the underlying pathogenesis to battle this dismal condition. We previously identified that AK4, a gene on chromosome 1p31.3 encoding a member of adenylate kinase family enzymes, was progressively up-regulated from normal colon, primary CRC to metastasis by mRNA gene expression microarray of paired CRC samples. This study was aimed to validate the overexpression of AK4 and explore in-vitro effects in CRC. We firstly examined the mRNA expression level of AK4 in a panel of CRC cell lines. AK4 mRNA expression was higher in CRC cell lines (n=8) than normal colon cell line, and significantly higher in advanced stage cell lines (n=4, Dukes’ C and D) than early stage ones (n=4, Dukes’ A and B) (P=0.03). We further evaluated expression level of AK4 in two different cohorts of clinical samples. The first cohort consisted of 18 paired primary CRCs and its corresponding normal colonic mucosa, and 27 metastatic CRC (including 15 cases with paired primary CRC and normal colonic mucosa). AK4 mRNA level was significantly higher in metastatic CRC than normal colon (P<0.001), and metastatic CRC than primary CRC (P<0.001). Among 15 cases with paired primary and metastatic tumors, 14 (93.3%) demonstrated higher AK4 in metastatic tumors than their primary counterparts(P<0.05). The second cohort composed of 190 consecutive primary CRCs. Upregulation of AK4 protein expression evaluated by immunohistochemistry was found in 68.4% of CRC, and associated with advanced AJCC stage (P<0.001), poorer overall survival (P=0.002) and worse disease-free survival (P<0.001). To evaluate functional properties of AK4 in CRC, we knocked down AK4 on a CRC cell lines with high endogenous AK4 expression (DLD1, HCT116 and SW620) by siRNA. knockdown of AK4 significantly reduced anchorage-dependent colony forming ability (P<0.05) and eliminated migratory (P<0.05) and invasive (P<0.05) abilities. In conclusion, AK4 is a potential oncogene upregulated in metastatic CRC and exhibiting in-vitro oncogenic properties. AK4 may play an important role in tumor progression and metastasis.

Citation Format: Yau Hei Yu, Joanna Hung Man Tong, Yi Pan, Raymond Wai Ming Lung, Ka Fai To, Anthony Wing Hung Chan. AK4 promotes colorectal cancer progression and 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 4858. doi:10.1158/1538-7445.AM2017-4858
Abstract 4863: Aurora-A kinase: a nuclear driver of metastasis
Authors: Marinak Kristina , Kiseleva Anna , Loskutov Yuriy , Smolkin Matthew , Pugacheva Elena
Published: July 2017

Aurora-A Kinase (AURKA) is a serine/threonine kinase that is critical for mitosis. AURKA is overexpressed in 96% of human cancers, including breast cancer. It has been previously shown that AURKA localizes to the nucleus in breast cancer metastases and especially in metastases of Triple Negative Breast Cancer (TNBC). Our objective is to define the role of nuclear AURKA in breast cancer metastasis in TNBC. Here we report that TNBC cells vary in amount of nuclear AURKA and this potentially correlates with their metastatic capabilities. Based on our preliminary findings we hypothesized that nuclear AURKA promotes cell survival and resistance to apoptosis in the metastatic niche. To test this hypothesis we created TNBC cell lines with CRISPR/Cas9 based deletion of endogenous AURKA. We also constructed exogenous AURKA specifically targeted to the nucleus by addition of a nuclear localization signal (NLS) or cytoplasm via addition of a nuclear exclusion signal (NES), respectively. To allow for in vitro and in vivo rescue experiments with exogenous NES or NLS AURKA in sgAURKA expressing TNBC cells, we introduced several silent mutations to avoid sgAURKA targeting. In our pilot orthotopic xenograft study with MDA-MB-231-luc2-Cas9 (TNBC) cells expressing sgAURKA with WT AURKA, NLS-AURKA or AURKA-NES shows a trend of increased metastatic colonization in the AURKA-NLS group. Overall, our results indicate that the amount of nuclear AURKA is increased in metastatic breast cancer cell lines and metastases of TNBC, but the mechanism of this translocation is currently unknown. Elucidation of this mechanism is critical for development of new therapeutical strategies for control and eradication of metastatic disease.

Citation Format: Kristina Marinak, Anna Kiseleva, Yuriy Loskutov, Matthew Smolkin, Elena Pugacheva. Aurora-A kinase: a nuclear driver of 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 4863. doi:10.1158/1538-7445.AM2017-4863
Abstract 4864: Role of AIB1 Δ -4 in malignant progression of breast cancer
Authors: Sharif Ghada M. , Ory Virginie , Wellstein Anton , Riegel Anna
Published: July 2017

The nuclear co activator AIB1 is amplified and/or overexpressed in up to 60 % of breast cancers. We have previously described a splice variant of AIB1 (Δ4) missing exon 4 that results in an N-terminally truncated protein. Full length AIB1 transcriptional function can be repressed by the tumor supressor ANCO1/ANKRD11. However, Δ4 is missing the ANCO1 binding domain and is not transcriptionally suppressed. More metastatic variants of isogenic breast or colon cancer cell lines have significantly increased levels of Δ4 relative to full length protein. Δ4 levels also increase in breast tissue samples as they transition in malignant progression. We conjectured that small increases in Δ4 protein in the cell could have potent effects on malignant progression. To determine the functional significance of Δ4 in malignant progression we developed DCIS (ductal carcinoma in situ) cell lines that only express this isoform and not the full length protein. We engineered these lines by using CRISPR technology targeted at the exon 3/4 splice junction. The resulting lines were significantly more invasive in ECIS endothelial invasion assays. In vivo, intracardiac zebrafish injections of the Δ4-DCIS lines showed higher extravasation from the tail vasculature compared to the parental line. Subcutaneous xenografts in athymic nude mice grew larger tumors, presented more invasive lesions and metastases. Also, Δ4-DCIS spheres growing in matrigel, showed disorganization and enhanced invasion into the matrix. In conclusion these data suggest a model where the increased splicing of AIB1 to the Δ4 variant alters transcription at specific target genes and contributes to the metastatic phenotype.

Citation Format: Ghada M. Sharif, Virginie Ory, Anton Wellstein, Anna Riegel. Role of AIB1 Δ -4 in malignant progression of 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 4864. doi:10.1158/1538-7445.AM2017-4864
Abstract 487: A comprehensive target enrichment panel for fusion detection
Authors: Saraiya Ashesh , Young Brandon , Meißner Tobias , Jones Brian L. , Huelga Stephanie C. , Amorese Doug A.
Published: July 2017

Growing interest in cancer classification and progression has accelerated the rate of novel gene fusions discovery with increasing recognition of their roles as biomarkers. RNA-Seq is an attractive method for expressed fusion discovery and detection because of its ability to provide unbiased fusion sequencing information. The ability to detect low expressing fusion transcripts, however, require high sequencing depth and represents a significant financial barrier and identification of clinically relevant fusion sequences from a large data set can be a bioinformatics challenge.

To address these challenges we have tested the Ovation® Fusion Panel Target Enrichment System V2, a targeted RNA sequencing method using the Single Primer Enrichment Technology (SPET), with a number of control and clinical samples. Initial studies were performed using a comprehensive target enrichment panel targeting 502 genes with three samples from Horizon DX containing known fusions. Target enriched libraries were constructed with 10 ng and 100 ng inputs and the data was analyzed using the NuFuseD pipeline (available as a point and click BaseSpace application or downloadable linux package) which has been optimized for fusion analysis from this data. Expected fusions were identified at both input levels, even when down sampled to 500K reads, with fewer fusion calls compared to other publically available fusion detection software (Chimerascan and SOAPFuse), suggesting a lower false positive rate. NuFuseD fusion calls are provided with a P-value to help prioritize the identified fusions for subsequent validation. Additionally, NuFuseD detected novel fusions in the control samples demonstrating the advantage of a comprehensive panel compared to more restricted panels. We further validated the target panel using control RNA (UHR and Human Brain) and fresh or FFPE cell lines (NCI-H2228, HCC1937) to further demonstrate our ability to identify known fusions.

Finally, the system was evaluated at an external site using patient FFPE samples. These samples (N=8) were from a set of breast, liver and ovarian cancers, containing a unique fusion in 4 of the samples based on DNA based sequencing. Only 1 of the 4 expected fusions were identified using whole transcriptome data (100 million reads) while 3 of the 4 fusions were detected with this assay (10 million reads) demonstrating its ability to generate targeted RNA sequencing libraries with increased sensitivity of gene fusion detection and reduced sequencing costs compared to standard RNA-Seq methods.

Citation Format: Ashesh Saraiya, Brandon Young, Tobias Meißner, Brian L. Jones, Stephanie C. Huelga, Doug A. Amorese. A comprehensive target enrichment panel for fusion detection [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 487. doi:10.1158/1538-7445.AM2017-487
Abstract 4873: Shedding light on the unknown of Ewing sarcoma: Single cell study shows a co-expression pattern between TAPA-1 and EWSR1-FLI1
Authors: Khoogar Roxane , Phelps Doris , Houghton Peter
Published: July 2017

Introduction: Ewing Sarcoma (ES) is the second most frequent bone cancer found in children, and has the highest rate of recurrence among all childhood cancers. This suggests the need for a better understanding of heterogeneous cancer populations and rare cells that survive intensive cytotoxic therapy and cause recurrence in patients. It is widely accepted that ‘EWSR1-FLI1’ has a major role in determining ES survival and proliferation, but the mechanism is unknown. Most studies in which expression of ‘EWSR1-FLI1’ is correlated with the level of expression of ‘EWSR1-FLI1’ regulated genes have been conducted on bulk populations that ignore potential heterogeneity of ‘EWSR1-FLI1’ in the cell population. To address this we have used single cell RNA-Seq approaches.

Methods: The WaferGen iCell8 system was used to perform SMART whole transcriptome amplification using EW-8 ES cells.We sequenced single-cell RNA-Seq libraries prepared from over 1260 ES parental cells. We combined computational analysis with stringent quality control filters to profile candidate ES cells.We used EWSR1 expression profile to partition the entire population into two clusters. A population of cells with TAPA-1 expression was distinguished and studied for their co-expression patterns with EWSR1.

Results: Results revealed that ES cells show considerable heterogeneity at the transcriptome level between seemingly identical EW-8 cells.

We identified a subpopulation of EW-8 ES cells with a positive correlation between the expression of EWSR1 and Target of the antiproliferative antibody 1 and tetraspanin-28 (TAPA-1/CD81). Our preliminary data from 200 cells indicate that EWSR1 (0-18 unique transcripts, median 1) and TAPA-1/Cd81 (0-8 unique transcripts, median 0) were correlated (r= 0.32, P<0.10). TAPA-1, a surface membrane protein, most frequently associated with hematopoetic cells, that regulates cell development, activation, growth and motility and has been implicated in proliferation of many cancers.

Conclusions: Taken together, our results suggest that there are two classes of cells with respect to the expression of TAPA-1 in EW-8 cells. We also show the relationship between TAPA-1 with EWSR1-FLI1 expression in a single cell level. This type of classification may lead us to identify a rare population with a poor response rate to cytotoxic treatments. Future studies will determine if EW-8 ES cells having elevated TAPA-1 have differential sensitivity to cytotoxic agents compared with populations with low level expression.

Citation Format: Roxane Khoogar, Doris Phelps, Peter Houghton. Shedding light on the unknown of Ewing sarcoma: Single cell study shows a co-expression pattern between TAPA-1 and EWSR1-FLI1 [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 4873. doi:10.1158/1538-7445.AM2017-4873
Abstract 4874: Application of quantitative analysis of mouse cell contamination and purification of human tumor cells in patient derived orthotopic xenograft mouse models of pediatric brain tumors
Authors: Braun Frank K. , Kogiso Mari , Qi Lin , Zhang Huiyuan , Du Yuchen , Lindsay Holly B. , Zhao Sibo , Injac Sarah G. , Li Xiao-Nan
Published: July 2017

Background: Pediatric brain tumors continue to be among the most difficult to cure and are the leading cause of cancer-related deaths in children. Research investigating pediatric brain cancer is still impaired by the lack of cancer models. We established a large panel of orthotopic xenograft models as well as several paired permanent cell lines using in vivo and in vitro culture techniques. However an inherent risk of xenograft models is the contamination of isolates of human tumors with host (mouse) cells. Therefor strategies to correctly and timely identify the human/mouse ratio are essential. In this study we investigated Prostaglandin Receptor EP2 (PTGER2) as a useful target to identifying mouse cell contamination in PDOX models.

Method: Species specific primers based on Alcoster et al., 2011 were used to detect PTGER2 in genomic DNA extracts of xenograft and cultured cells. Real-time qPCR was carried out using a StepOnePlus™ Real-Time PCR System. Reaction mix contained SYBR Green select master mix, respective primers and 5 ng of total genomic DNA in a total reaction volume of 10 μL. The qPCR conditions were as follows: 50°C-2 min, 95°C-10 min, 40 cycles of (95°C-15 sec, 61°C-1 min). Primer specificity was controlled in addition by analysis of amplified products on a 1.2% agarose gel. FACS was used to isolate human cells according to anti-Human HLA-ABC Ab and mouse-specific Ab cocktail surface staining and subjected to qPCR.

Results: Various models of pediatric brain cancer (PXA, MB, GBM, EPN, ATRT, DIPG, PNET) established in our group were investigated for mouse cell contamination. Cells from xenograft isolates as well as from cultured xenografts were routinely analyzed. Our study confirms PTGER2 as a useful target for accurately estimate mouse cell contamination in brain cancer xenograft models. This further enabled us to improve detection and characterization of HLA-ABC/mouse-cocktail double negative population as seen in FACS.

Conclusion: In conclusion we have successfully implemented an easy qRT-PCR approach to determine the human/mouse cell ration in any mixed cell population. Particular for PDOX models it is paramount to have reliable information on mouse cell contamination.

Citation Format: Frank K. Braun, Mari Kogiso, Lin Qi, Huiyuan Zhang, Yuchen Du, Holly B. Lindsay, Sibo Zhao, Sarah G. Injac, Xiao-Nan Li. Application of quantitative analysis of mouse cell contamination and purification of human tumor cells in patient derived orthotopic xenograft mouse models of pediatric brain tumors [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 4874. doi:10.1158/1538-7445.AM2017-4874
Abstract 4895: CT20p as a therapeutic for lung cancer with elevated chaperoning containing TCP1 (CCT) expression levels
Authors: Carr Ana C. , Khaled Amr S. , Bassiouni Rania , Khaled Annette R.
Published: July 2017

Lung cancer is the leading cause of cancer related fatalities worldwide and although there is a substantial effort in the development of improved, more specific, and less debilitating therapies, very little progress has been made accomplishing this goal. We propose the use of CT20p, a therapeutic peptide developed by our laboratory, as novel therapy for lung cancer. We have previously demonstrated that CT20p has tumor specific cytotoxicity in breast cancer cells and it induces tumor regression in xenograft models of breast cancer. We have also determined Chaperonin containing T-complex (CCT) to be the intracellular target of CT20p and that susceptibility to CT20p correlates with its expression levels, with tumor cells expressing high levels of CCT being the most susceptible. CCT is a large macromolecular complex composed of 8 subunits (CCTα, CCTβ, CCTγ, CCTδ, CCTϵ, CCTζ, CCTη, CCTθ). Immunoblot probing against four relevant CCT subunits (CCTβ, CCTγ, CCTδ, CCTϵ) was used to determine their relative expression level in five small cell lung cancer (SCLC) cell lines when compared to normal lung cells. We determined that the expression levels of the four probed CCT subunits varied amongst the five SCLC cell lines, however, the four CCT subunits were always expressed at higher levels when compared to normal lung cells. Immunohistochemistry staining for CCTβ was used on tissue microarray (TMA) of lung cancer progression. Results were interpreted on a scale of 1 to 4 (with 4 being the strongest staining). We determined that CCTβ levels increase with cancer severity and identified a significant correlation between CCTβ levels and cancer progression in both small cell lung carcinoma (SCLC) and Squamous cell lung carcinoma (SqCLC). Using The Cancer Genome Atlas (TCGA) database, we have found CCT gene alterations in clinical lung cancer cases range from 10% to 43%, however, only CCT4 gene amplification in SqCLC cases correlated with decreased survival. Altogether, these results suggest that CT20p has the potential for being a therapeutic for multiple types of lung cancer due to increased target expression.

Citation Format: Ana C. Carr, Amr S. Khaled, Rania Bassiouni, Annette R. Khaled. CT20p as a therapeutic for lung cancer with elevated chaperoning containing TCP1 (CCT) expression levels [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 4895. doi:10.1158/1538-7445.AM2017-4895
Abstract 4907: Elucidating the inhibitory role of plant alkaloids in breast cancer metastasis
Authors: Iqbal Waqas , Alkarim Saleh , Sabir Jamal S. , Bora Roop S. , Saini Kulvinder S.
Published: July 2017

Despite extremely high morbidity and mortality of >90% from metastasis, it remains one of the most challenging process for targeted therapeutic interventions. Identification and characterization of chemically active and bioavailable compounds from medicinal plants targeting the cell-signaling pathways of Cancer Stem Cells (CSCs), tumor cells and their microenvironment that abet metastasis is of great clinical significance. Hence, MCF-7; a breast cancer cell line, was treated with fractionated alkaloidal extracts from Rhazya stricta with previously documented anti-cancer activity in lung and pancreatic cancer cell lines. RNA-seq. analysis was performed for samples treated with compounds that actively inhibited cell migration in scratch assay to further substantiate their anti-metastatic role at the cellular and molecular level. Genomic data corroborated the presence of anti-metastatic compounds that actively down-regulated genes involved in promoting metastasis (CDC6), cell migration (KIF23, DLGAP5), invasion and CSCs’ expansion (PTTG1). We also found down-regulation of GAS1 expression, an enhancer of Hedgehog signaling pathway. To our knowledge, this study, first of its kind, sets precedence in exploiting the anti-metastatic potential of these compounds. Further proof of concept studies in mouse models of breast cancer to establish precise mechanism-of-action(s), would certainly open up avenues for the development of new chemical entities (NCEs), targeting metastasis regulating gene(s) with precision.

Citation Format: Waqas Iqbal, Saleh Alkarim, Jamal S. Sabir, Roop S. Bora, Kulvinder S. Saini. Elucidating the inhibitory role of plant alkaloids in 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 4907. doi:10.1158/1538-7445.AM2017-4907
Abstract 4910: Eribulin rapidly impairs TGF-β signaling
Authors: Kaul Roma , Risinger April L. , Mooberry Susan L.
Published: July 2017

Microtubule targeting agents (MTAs) continue to be valuable in treating breast cancer. While decades of research have shown that these drugs cause mitotic arrest in cells by suppressing the dynamic instability of microtubules, recent evidence demonstrates that the ability of MTAs to disrupt the microtubule-dependent transport of key signaling components in interphase cells likely contributes to their anticancer actions. Cell signaling messages are relayed by proteins that organize or scaffold signaling hubs. These signaling nodes facilitate the complex organization and coordination of multiple signaling partners. NEDD9 is a member of the CAS scaffold family and it has been shown to coordinate TGF-β signaling, a driver of oncogenesis and epithelial-to-mesenchymal transition (EMT). Ligand-mediated stimulation of TGF-β receptors leads to the activation of downstream canonical and non-canonical signaling pathways. These pathways collectively induce the expression of Snail and Slug, key transcriptional-repressors that promote EMT. We tested the hypothesis that a short-term treatment of breast cancer cells with eribulin or 3 other clinically relevant MTAs would differentially disrupt interphase microtubules and alter TGF-β-dependent signaling. BT-549 cells were treated for 2 h with concentrations of MTAs that are clinically relevant and cause maximum disruption of the interphase microtubule network; 100 nM was used for the destabilizers, eribulin and vinorelbine and 1 µM was used for the stabilizers, paclitaxel and ixabepilone. The results showed that eribulin and vinorelbine significantly inhibited TGF-β- induced expression of Snail and Slug. The microtubule stabilizers had no effect on Snail and Slug expression following TGF-β stimulation. Further studies evaluated whether NEDD9 contributes to the downregulation of Snail and Slug. Co-immunoprecipitation and knockdown studies suggest that eribulin impairs the ability of NEDD9 to scaffold TGF-β signaling partners, thus inhibiting downstream transcriptional signaling. Consistent with the ability of eribulin to reverse EMT in experimental models within 7 days, this study begins to shed light on the mechanisms underlying its action. Funding for this work was provided by Eisai Inc.

Citation Format: Roma Kaul, April L. Risinger, Susan L. Mooberry. Eribulin rapidly impairs TGF-β signaling [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 4910. doi:10.1158/1538-7445.AM2017-4910
Abstract 4912: Effect of the combined treatment with mifepristone and chemotherapy on breast cancer brain metastases
Authors: Rubin Ayelen , Fuente Veronica de la , Salvatierra Edgardo , Vanzulli Silvia , Podhajcer Osvaldo , Lanari Claudia , Rojas Paola
Published: July 2017

The treatment of brain metastasis is limited due to the drug’s inability to cross the blood brain barrier, because of the presence of endothelial cells’ multidrug resistance efflux transporters, such as the P-glycoprotein (P-gp). The antiprogestin Mifepristone (MFP) is known to inhibit the P-gp activity. We hypothesize that a combination of MFP and Pegylated doxorubicin liposomes (doxo) improves the drug efficacy on breast tumors that do not express progesterone receptors. Using the metastatic triple negative MDA-231-BrM2 breast cancer cell line, transfected with GFP and luciferase, we explored different protocols to generate tumors growing in the brain of NSG mice. Spontaneous metastases were obtained by subcutaneous injection (sc) of 2 x 106 cells, and experimental metastases by intracardiac injection (ic) of 1 x 106 cells or by intracranial injection of 2 x 105 cells. In the ic and sc models small and scattered brain metastases as well as liver, lung and kidney metastases were observed. The intracranial model enabled us to obtain measurable brain tumors in a short time. Thus we selected this method to evaluate the effect of MFP (6 mg pellets, sc) and /or doxo (4,5 mg/kg, iv) on tumors growing in the brain. Treatment was initiated 10 days after cell inoculation. The amount of tumor cells after brain excision was measured by flow cytometry (GFP) and by luminometry (luciferase activity). A decreased luminescence signal was observed in brains from mice treated with both agents as compared with the control mice (p<0.05), whereas no effects were observed between the single treatment groups and the controls. Moreover, the flow cytometry analysis showed that the combined treatment, MFP+doxo, reduced the number of tumor cells further than the single chemotherapeutic agent (p<0.05). Our data suggest that MFP may be a promising agent to treat brain metastasis of breast cancers, which do not necessarily express progesterone receptors, to increase the effectiveness of chemotherapeutic agents. The participation of the P-gp mediating this effect needs to be confirmed.

Citation Format: Ayelen Rubin, Veronica de la Fuente, Edgardo Salvatierra, Silvia Vanzulli, Osvaldo Podhajcer, Claudia Lanari, Paola Rojas. Effect of the combined treatment with mifepristone and chemotherapy on breast cancer brain metastases [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 4912. doi:10.1158/1538-7445.AM2017-4912
Abstract 4917: Therapeutic inhibition of Notch1 in metastatic prostate cancer
Authors: Rice Meghan A. , Hsu En-Chi , Stoyanova Tanya
Published: July 2017

Metastatic castration-resistant prostate cancer (CRPC) is the primary cause of prostate cancer specific mortality, and is currently incurable. Defining new mechanisms that can predict progression and drive lethal CRPC is critical. Notch1 receptor, a cell surface-signaling molecule dysregulated in many cancers, is highly expressed in CRPC. Chronic activation of Notch1 synergizes with multiple oncogenic pathways altered in early disease to promote the development of poorly differentiated prostate carcinoma. Consistent with its activation in clinical CRPC, tumors driven by NICD1 in combination with alternative pathways altered in prostate cancer are metastatic and resistant to androgen deprivation.

Genetic and pharmacological inhibition of Notch1 activity decreases cell proliferation and colony formation of 22RV1 prostate cancer cells in vitro. 22RV1 subcutaneous xenograft growth was significantly impaired by treatment with pharmacological inhibitors of Notch1 activation over the course of 20-day treatment. Further, Notch1 inhibition in combination with other standard prostate cancer treatments compounded efficacy of individual treatments to attenuate colony formation potential of prostate cancer cells in vitro.

The Notch1 signaling axis, confirmed to synergize with multiple pathways to promote metastatic CRPC already provides a novel pathway for diagnostic stratification of prostate cancer patients. Our studies additionally implicate Notch1 as a therapeutically relevant target for aggressive prostate cancer.

Citation Format: Meghan A. Rice, En-Chi Hsu, Tanya Stoyanova. Therapeutic inhibition of Notch1 in metastatic prostate 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 4917. doi:10.1158/1538-7445.AM2017-4917
Abstract 4929: Cooperation between the microbiota and oncogenic mutations leading to the development of colon cancer
Authors: Hey Dana Van De , Payne Susan , Pasch Cheri , Clipson Linda , Matkowskyj Kristina , Deming Dustin
Published: July 2017

Background: The microbiome of the colon has been shown to play a role in colon cancer tumorigenesis. Inflammation is a key step in the formation of tumors, and it is possible that microbiota induce this inflammation, further stimulating tumor growth. The interaction between mutant cells in the colon and the microbiota has been understudied. We hypothesized that eradication of the intestinal microbiota could prevent tumorigenesis in the colon despite the presence of oncogeneic mutations.

Methods: We have previously generated a transgenic murine model in which a constitutively active PI3K is expressed in the distal small intestine and colon (FC3K). These mice develop aggressive cancers in the proximal colon via a non-canonical mechanism without loss of APC. In addition, we developed a murine model possessing both PIK3CA mutations and loss of APC (FCAK3K). These mice were treated with vancomycin in their drinking water. Necropsy was performed at specific time-points or at moribund to determine tumor counts and size. Spheroid cultures were also utilized to determine the potential for cytotoxic effects of vancomycin treatment.

Results: Treatment with vancomycin in FC3K mice at weaning resulted in a reduction in tumor incidence and growth and prolonged survival, indicated by a 42% tumor frequency and median lifespan of 101 days, compared to 100% frequency and 86.5-day lifespan of control mice (p<0.001). In the tumors that formed there was no difference in the epithelial mesenchymal transition (EMT), Ki67 index, nuclear localization of CTNNB1, or activation of the PI3K/AKT/mTOR signaling pathway. FCAK3K spheroid and mouse models portrayed ineffectiveness of vancomycin through an absent change in spheroid diameter and also 100% frequency of both treated and control mice.

Conclusion: The above results indicate that colon cancers induced by PIK3CA mutations are at least partially dependent on the microbiota for tumorigenesis. This dependence appears to be overcome in the setting of both APC and PIK3CA mutations.

Citation Format: Dana Van De Hey, Susan Payne, Cheri Pasch, Linda Clipson, Kristina Matkowskyj, Dustin Deming. Cooperation between the microbiota and oncogenic mutations leading to the development of colon 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 4929. doi:10.1158/1538-7445.AM2017-4929
Abstract 4934: Brain metastatic microenvironment reshapes cancer cell metabolism through epigenetic up-regulation of glutamate decarboxylase 1
Authors: Schnepp Patricia M. , Lee Dennis D. , Guldner Ian H. , O'Tighearnaigh Treasa , Palakurthi Bhavana , Eckert Kaitlyn E. , Toni Tiffany A. , Ashfeld Brandon L. , Zhang Siyuan
Published: July 2017

Cancer metabolism has been well documented to influence primary tumor development. Yet, the role of deregulated metabolism in metastasis progression is not well understood. Based on a distinct metabolic transcriptome profile in brain metastases compared to their primary tumor counterparts, we hypothesized that metabolic transcriptome shifting during metastatic evolution is crucial for metastatic success to the brain. Here we show that, despite a global down-regulation of multiple metabolic pathways in the brain metastatic tumors, the expression of the GABA metabolic pathway mediator glutamate decarboxylase 1 (GAD1) is significantly up-regulated. Using cell based co-culture models for different primary and brain metastatic microenvironments and in vivo brain metastasis models, we demonstrate that down-regulation of DNA methyltransferase 1 (DNMT1) induced by the brain microenvironment results in decreased GAD1 promoter methylation and subsequent up-regulation of GAD1 expression in tumor cells. To dynamically visualize the cellular metabolic responses mediated by GAD1, we utilized the Peredox biosensor to monitor the cytosolic NADH:NAD+ equilibrium in tumor cells using time-lapse imaging. By knocking down of GAD1 induced by primary glia cell co-culture, we abolished the capability of tumor cells to utilize extracellular glutamine, leading to an NADH accumulation in the cytosol and a more oxidative cellular status. Lastly, either loss of GAD1 genetically or targeting GABA metabolic pathway by the repurposing of a neurological drug, vigabatrin, results in a significant decrease in brain metastasis incidence. Taken together, our results demonstrated that brain microenvironment-specific metabolic shifting through GAD1 promoter de-methylation drives brain metastasis outgrowth.

Citation Format: Patricia M. Schnepp, Dennis D. Lee, Ian H. Guldner, Treasa O'Tighearnaigh, Bhavana Palakurthi, Kaitlyn E. Eckert, Tiffany A. Toni, Brandon L. Ashfeld, Siyuan Zhang. Brain metastatic microenvironment reshapes cancer cell metabolism through epigenetic up-regulation of glutamate decarboxylase 1 [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 4934. doi:10.1158/1538-7445.AM2017-4934
Abstract 4946: Host tissue stiffness regulates chemotherapy-induced cancer cell dormancy
Authors: Anlas Alisya , Nelson Celeste M.
Published: July 2017

This study investigates how the tumor microenvironment, namely host tissue stiffness, affects the propensity of disseminated tumor cells to become dormant at secondary sites.

Approximately 90% of cancer-related deaths result from metastasis. In breast cancer, disseminated cancer cells cannot be detected at the time of diagnosis of the primary tumor because they form “dormant” micrometastases that are not clinically detectable. Thus, metastases in secondary sites such as the lungs, bone marrow, liver and brain are

usually discovered months or years after the initial diagnosis and/or treatment of the primary tumor.

A 2D polyacrylamide gel-based tissue culture model was used to investigate the effects of host tissue stiffness on the proliferative behavior of breast cancer cells. To assess the dormant phenotype, Ki67 and RNA staining were used to quantify proliferation and RNA

content, respectively. Additionally, changes in levels of cell-cycle regulators were determined by quantitative RT-PCR. Dormant cells have decreased levels of transcription and proliferation.

We found that both tamoxifen and 5-fluorouracil induce dormancy in estrogen receptor-positive breast cancer cells cultured on soft and stiff substrata. After repeated rounds of 5-fluorouracil treatment and recovery, cells cultured on soft substratum, mechanical properties of which represent common metastatic sites, had a higher tendency to recover.

This suggests that cell growth at secondary sites favors a soft microenvironment, characteristic of organs where breast cancer metastases often become clinically overt. Dormant cancer cells, or minimal residual disease, cannot be detected with current

diagnostic tools and cannot be targeted by conventional therapies. Therefore, current clinical practices rely on estimating the probability of recurrence from various prognostic factors including the grade and the stage of the disease. Elucidating the mechanisms that regulate the switch from dormancy to proliferation at metastatic sites will pave the way for new treatments to control this incurable disease.

Citation Format: Alisya Anlas, Celeste M. Nelson. Host tissue stiffness regulates chemotherapy-induced cancer cell dormancy [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 4946. doi:10.1158/1538-7445.AM2017-4946
Abstract 4948: Characterization of hTERT-immortalized prostate-derived stromal and epithelial cells: An authentic in vitro model for tumor microenvironment studies
Authors: Rodriguez Luis G. , McDaniel Russell E. , Zhao Xiangshan , Turner Elizabeth , Annesi Christopher , Zou Chaozhong
Published: July 2017

Tumor development begins with mutational changes to the genetic makeup of a cell, but its progression is not solely determined by the mutated cell, but also by the tumor’s microenvironment. Prostate cancer, a leading cancer diagnosed in men, has been shown to be highly influenced by its surrounding stroma, particularly fibroblasts. It has been demonstrated that cancer-associated prostate fibroblast (CAFs) differ from normal-associated prostate fibroblast (NAFs). However, human prostate cancer model systems have focused largely on prostate cancer epithelial cells. Currently, a need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumor microenvironment. In this study, we characterized three prostate-derived cells: prostate cancer-associated fibroblast (CAFs), prostate normal-associated fibroblast (NAFs) and prostate cancer epithelial (PrE) cells; all three lines were immortalized by hTERT (human telomerase reverse transcriptase) alone, and have been continuously passaged for more than 40 PDL in our hands. Our data shows that the hTERT immortalized CAFs proliferate faster than the NAFs; in addition, both CAFs and NAFs express fibroblast markers such as TE7 and alpha smooth muscle actin (α-SMA), while neither cell line expresses epithelial marker such as CK14. Both CAFs and NAFs also express elevated levels of α-SMA upon TGF-β stimulation. All three prostate-derived cells express the prostate specific marker AR, and show similar markers staining after long time passaging. Importantly, conditioned media collected from CAFs promotes tumor cell growth better than NAF conditioned media. In conclusion, CAFs, NAFs, and immortalized prostate cancer epithelium may provide a very valuable model system for the study of prostate cancer cell progression and tumor microenvironment studies.

Citation Format: Luis G. Rodriguez, Russell E. McDaniel, Xiangshan Zhao, Elizabeth Turner, Christopher Annesi, Chaozhong Zou. Characterization of hTERT-immortalized prostate-derived stromal and epithelial cells: An authentic in vitro model for tumor microenvironment studies [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 4948. doi:10.1158/1538-7445.AM2017-4948
Abstract 4955: PD-L1 expression on circulating epithelial tumor cells (CETCs) correlates with the presence of metastasis in breast cancer patients and differs from PD-L2 expression
Authors: Pizon Monika , Schott Dorothea , Pachmann Ulrich , Pachmann Katharina
Published: July 2017

Background: Strategies to improve the efficacy of the immune system against malignant tumors represent a major innovation focusing on the programmed cell death-1 receptor (PD-1), with its two ligands PD-L1 and PD-L2. The expression of PD-L1 has been evaluated in a number of different tumor types and can be used as a predictive biomarker for PD-1/PD-L1 checkpoint inhibitor treatment response. PD-L2 has not received as much attention and its role in modulating tumor immunity is less clear. We used a non-invasive, real-time liquid biopsy to better characterize PD-L1/-L2 expression on circulating epithelial tumor cells (CETCs) in breast cancer patients.

Methods: CETCs were determined from blood of 72 patients suffering from breast cancer. The number of vital CETCs and their expression of PD-L1 and PD-L2 were investigated using the maintrac® method.

Results: PD-L1 expressing CETCs were detected in 94.5 % of breast cancer patients whereas only 82% patients had PD-L2 positive CETCs. Breast cancer patients with metastatic disease had significantly more PD-L1 positive CETCs as compared to patients without metastasis (median 75% vs. 61.1%; p<0.05). The fraction of PD-L1 positive CETCs was significantly higher than the fraction of PD-L2 positive CETCs (54.6% vs. 28.7%; p<0.001). Moreover, we observed a significant heterogeneity in PD-L1 and PD-L2 immunostaining intensity across CETCs from the same patients.

Conclusion: Breast cancer patients have detectable CETCs with a high frequency of PD-L1 which correlates with progression of cancer disease. PD-L1 seems to be a major factor in immune evasion and may be a promising target of anticancer therapies. Monitoring the frequency of PD-L1 positive CETCs could reflect individual patient’s response to an anti-PD-1/PD-L1 therapy.

Citation Format: Monika Pizon, Dorothea Schott, Ulrich Pachmann, Katharina Pachmann. PD-L1 expression on circulating epithelial tumor cells (CETCs) correlates with the presence of metastasis in breast cancer patients and differs from PD-L2 expression [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 4955. doi:10.1158/1538-7445.AM2017-4955
Abstract 4964: Structure, in vitro biology and in vivo pharmacodynamic characterization of a novel clinical IDO1 inhibitor
Authors: Hunt John T. , Balog Aaron , Huang Christine , Lin Tai-An , Lin Tai-An , Maley Derrick , Gullo-Brown Johnni , Swanson Jesse , Brown Jennifer
Published: July 2017

The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the degradation of tryptophan along the kynurenine pathway, and is frequently expressed in human malignancies. The activity of IDO1 induces an immunosuppressive microenvironment in tissues by inhibiting T-cell function through local depletion of tryptophan and through generation of kynurenine pathway metabolites. Inhibition of IDO1 is expected to diminish the immunosuppressive tumor microenvironment and improve cancer patient outcomes, particularly when used in combination with cancer immunotherapy agents such as nivolumab and ipilimumab. In this presentation, we will disclose the chemical structure, enzyme inhibitory mechanism, in vitro potency and in vivo pharmacodynamic (PD) activity of BMS’ IDO1 inhibitor currently in Phase I clinical trials. The compound is a potent and selective IDO1 inhibitor with no activity against another tryptophan degrading enzyme, tryptophan 2,3-dioxygenase (TDO). It exhibited potent cellular activity, suppressing kynurenine production in HEK293 cells overexpressing human IDO1 (IC50 = 1.1 nM) and in HeLa cells stimulated with IFNγ (IC50 = 1.7 nM). The compound also potently restored T-cell proliferation in a co-culture of T cells and human cancer cells and in a mixed lymphocyte reaction where T cells were co-cultured with allogeneic IDO1-expressing dendritic cells (EC50 = 1.2 nM). In vivo, when given once a day orally, the compound exhibited significant PD activity in mouse tumors grown subcutaneously in syngeneic hosts and in human tumors grown as xenografts in nude mice.

Citation Format: John T. Hunt, Aaron Balog, Christine Huang, Tai-An Lin, Tai-An Lin, Derrick Maley, Johnni Gullo-Brown, Jesse Swanson, Jennifer Brown. Structure, in vitro biology and in vivo pharmacodynamic characterization of a novel clinical IDO1 inhibitor [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 4964. doi:10.1158/1538-7445.AM2017-4964