bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-317: Interactive pipeline for reproducible genomics analyses
Authors: Considine Michael , Parker Hilary S. , Wei Yingying , Xia Xiao X. , Cope Leslie , Ochs Michael F. , Fertig Elana J.
Published: May 2015

Automated Genomics Analysis (AGA) is a program to analyze high-throughput genomic data sets, such as Affymetrix gene expression arrays. The software implements an easy to use, point and click, guided pipeline to combine, define and compare datasets, and customize their outputs. Batch correction techniques are implemented to combine data sets. Options to save plots, tables and data are available throughout the analysis, and log files containing the R script run are also generated to facilitate reproducible analyses.

The software has been applied to gene expression measurements from head and neck squamous cell carcinoma (HNSCC). It has batch corrected several public domain microarray HNSCC datasets, facilitating accurate comparison of subtypes such as HPV status. Moreover, current extensions enable users to interactively perform reproducible genomics analysis using clinical covariates in RNA sequencing data from TCGA.

Citation Format: Michael Considine, Hilary S. Parker, Yingying Wei, Xiao X. Xia, Leslie Cope, Michael F. Ochs, Elana J. Fertig. Interactive pipeline for reproducible genomics analyses. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-317. doi:10.1158/1538-7445.AM2014-LB-317
Abstract LB-319: Functionally validated shRNA libraries for large-scale RNAi screens
Authors: Sridhar Vaishali , Lai Dan Yu , Sinha Nishi , Premsrirut Prem K. , Fellmann Christof
Published: May 2015

RNA interference (RNAi) has become an indispensable tool for loss-of-function genetics across eukaryotes. By enabling stable and reversible gene silencing, short hairpin RNA (shRNA) triggers of RNAi provide a means to study long term phenotypes, perform pool-based forward genetic screens and examine the consequences of temporary target inhibition in vivo. However, efficient implementation in vertebrate systems has been hindered by our incomplete understanding of the sequence requirements for effective shRNA biogenesis and target suppression. Consequently, single-gene studies still depend on laborious testing of many candidates and pooled shRNA screens contain non-functional sequences that make the interpretation of negative results inconclusive.

To address this limitation, we recently developed a high-throughput “RNAi Sensor assay” that enables functional identification of effective shRNAs at large scale. We showed that this assay allows testing of tens of thousands of candidates in parallel and robustly identifies potent single-copy shRNAs targeting virtually any gene. Here we applied this technology to evaluate 20,000 preselected candidate shRNAs targeting over 330 disease-relevant mouse genes to generate a functionally validated shRNA library for potent single-copy target knockdown. Beyond their use in RNAi transgenic animals, such shRNA libraries will enable comprehensive pooled RNAi screening and open a promising avenue for high-throughput functional genetics in the post-genomic era.

Citation Format: Vaishali Sridhar, Dan Yu Lai, Nishi Sinha, Prem K. Premsrirut, Christof Fellmann. Functionally validated shRNA libraries for large-scale RNAi screens. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-319. doi:10.1158/1538-7445.AM2014-LB-319
Abstract LB-37: A patient-derived in vitro model of colorectal cancer: a perfusion culture system that recapitulates patient tumor composition and structure in 3-dimensions
Authors: Pfund William P. , Neeb Paul A.
Published: May 2015

Cancer medicine would benefit greatly from a practical technology that accurately predicts the sensitivity of individual patient tumors to drugs. Previously developed in vitro chemosensitivity models have generally failed to do this because they do not incorporate key aspects of the tumor microenvironment (TME), which clearly plays a role in drug resistance. There has been significant interest in patient-derived xenograft (PDX) chemosensitivity testing as an alternative to in vitro models, but the applicability of this approach is limited by engraftment percentages and growth rates.

We have developed an in vitro technology that is designed to recapitulate the 3D tissue architecture of patient-derived colorectal tumors including accessory (endothelial and immune) cells, starting with freshly excised tumor specimens. The system incorporates: 1) low-sheer, tangential flow of nutrient medium above and below an open, synthetic 3D cell scaffold; and 2) exchange of metabolic gasses via permeable membranes which separate the culture chamber core from gassing chambers located above and below the nutrient flow compartments. This design allows establishment of gas and nutrient gradients across the cell scaffold that closely mimic conditions in vivo.

Aliquots of mechanically and enzymatically dissociated, freshly excised, primary human colorectal adenocarcinomas were introduced into the 3D culture system and maintained for up to 3 weeks. Routine maintenance of the cultures involved partial medium exchanges as needed based on glucose concentrations in the circulating medium. The proportions and arrangement of key cell types (e.g., normal muscularis, tumor epithelium, stromal fibroblasts, endothelial cells, and immune cells) in the cultured explants were compared to those of the original tumor by standard histological techniques. Results revealed good agreement between the histology of the original tumor and that of the in vitro model. These results suggest that this model technology could be applied to individual patient drug selection as well as studies of colorectal cancer biology that are not feasible or practical in vivo.

Citation Format: William P. Pfund, Paul A. Neeb. A patient-derived in vitro model of colorectal cancer: a perfusion culture system that recapitulates patient tumor composition and structure in 3-dimensions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-37. doi:10.1158/1538-7445.AM2014-LB-37
Abstract LB-39: Human cone precursor-derived retinoblastoma tumors
Authors: Xu Xiaoliang L. , Singh Hardeep P. , Wang Lu , Qi Donglai , Poulos Bradford K. , Abramson David H. , Jhanwar Suresh C. , Cobrinik David
Published: May 2015

Retinoblastomas initiate in response to biallelic RB1 inactivation and loss of functional Rb protein, yet the cell of origin and cellular circuitry that sensitizes to Rb loss have been unclear. Here, we probed the cellular origin of retinoblastoma by assessing cell type-specific responses to shRNA-mediated Rb depletion in dissociated fetal retina, in intact retina, and in isolated cone precursor populations. In dissociated retinal cultures, RB1 knockdown induced S phase entry and proliferation of cells expressing markers of cone precursors (CRX + L/M opsin, or CRX + cone arrestin, or CRX + RXRγ) but not markers or other retinal cell types. Similarly, RB1 knockdown induced cone precursor proliferation in the undissociated retina and in prospectively isolated cone precursor populations. Proliferation of FACS-isolated cone precursors correlated with the induction of E2F-responsive genes and depended upon factors having strong cone precursor expression and crucial roles in retinoblastoma cell proliferation, including MDM2, MYCN, TRβ2, and SKP2. The proliferative response to Rb depletion was enhanced by co-depletion of the Rb-related p130 but was abrogated by co-depletion of p107, concordant with frequent p130 but not p107 losses in retinoblastoma tumors. Rb- and Rb/p130-depleted cone precursors proliferated in suspension and formed tumors in orthotopic xenografts with histologic features and protein expression profiles typical of differentiated human retinoblastomas. These findings indicate that post-mitotic human cone precursors are uniquely sensitive to the proliferative and oncogenic effects of Rb depletion and imply that human cone precursor circuitry collaborates with inactivating RB1 mutations to initiate retinoblastoma tumorigenesis.

Citation Format: Xiaoliang L. Xu, Hardeep P. Singh, Lu Wang, Donglai Qi, Bradford K. Poulos, David H. Abramson, Suresh C. Jhanwar, David Cobrinik. Human cone precursor-derived retinoblastoma tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-39. doi:10.1158/1538-7445.AM2014-LB-39
Abstract LB-41: Prolonged survival upon ultrasound-enhanced doxorubicin delivery in two syngenic glioblastoma mouse models
Authors: Kovaks Zsofia , Werner Beat , Martin-Fiori Ernst , Bernasconi Michele
Published: May 2015

Glioblastoma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor, with a very poor prognosis. Chemotherapeutical treatment of GBM is limited by the blood-brain barrier (BBB). This physical and metabolic barrier separates the blood from the brain parenchyma and prevents the entry of toxins but also of potentially useful chemotherapeutics from the blood into the brain.

Microbubble-enhanced focused ultrasound (MB-FUS) has been proposed to disrupt locally and reversibly the BBB to facilitate diffusion of drugs from the micro vasculature into brain tissue. The present study investigates the feasibility and the safety of such an approach in two syngenic mouse models of GBM (GL261 and SMA-560).

Local doxorubicin (DOX) concentration in MB-FUS sonicated normal brain tissue as well as in brain tumor tissue was increased as compared to the unsonicated control tissue in the contralateral hemisphere. Moreover, ultrasound mediated BBB disruption, in combination with DOX therapy, resulted in a significant increase of survival and in a slower disease progression in the two syngenic GBM mouse models.

In conclusion, our results confirm that MB-ultrasound might ultimately be an effective technology to improve the therapy of GBM, and they provide for the first time evidence that combining MB-FUS with DOX treatment is effective in syngenic mouse models for GBM which can serve as preclinical models to study the impact of immune system on the therapeutic application of MB-FUS chemotherapy.

Citation Format: Zsofia Kovaks, Beat Werner, Ernst Martin-Fiori, Michele Bernasconi. Prolonged survival upon ultrasound-enhanced doxorubicin delivery in two syngenic glioblastoma mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-41. doi:10.1158/1538-7445.AM2014-LB-41
Abstract LB-48: DCLK1 targeted monoclonal antibodies demonstrate therapeutic potential against pancreatic ductal adenocarcinoma
Authors: Qu Dongfeng , Weygant Nathaniel , May Randal , Chandrakesan Parthasarathy , Owen Daniel , Sureban Sripathi , Houchen Courtney
Published: May 2015

Doublecortin-like kinase 1 (DCLK1) is overexpressed and marks a population of tumor-initiating cells in pancreatic ductal adenocarcinoma (PDAC). It regulates key oncogenes, pluripotency factors, angiogenic factors, and epithelial mesenchymal transition (EMT) related transcription factors. Therefore, DCLK1 may be a novel therapeutic target in PDAC. In this study we evaluated whether DCLK1-targeted monoclonal antibodies (mAbs) demonstrate therapeutic activity against pancreatic cancer. Human pancreatic cancer cells (AsPC-1) were treated with mAbs against the C-terminal domain of human DCLK1 to assess their effect on gene expression, cell proliferation, migration, invasion, and tumorigenicity. Gene expression levels were analyzed by quantitative real-time RT-PCR. The proliferative and invasive potential of cells with or without mAb treatment were compared using a MTT assay for proliferation, wound healing assay for migration, and Matrigel coated transwell assay for invasion. Tumor xenografts originated from AsPC-1 cells in nude mice were also treated with these mAbs. Here we demonstrate that treating AsPC-1 cells with mAb against DCLK1 resulted in a more than 50% decrease in DCLK1 mRNA expression. The expression levels of several key tumor suppressor microRNAs, let7a, miR144, and miR200, were upregulated in cells treated with these mAbs. The expression levels of pancreatic cancer related oncogenes KRAS, NOTCH, and REG4, angiogenic factors, VEGFR1 and VEGFR2, and EMT related genes, ZEB1, ZEB2, SNAIL, SLUG, and TWIST, were significantly downregulated (among 30-50% reduction) in the cells treated with mAb against DCLK1. Furthermore, AsPC-1 cells exhibited more than 20% decrease in migration and more than 40% decrease in invasion following anti-DCLK1 mAb treatment. Moreover, AsPC-1 xenograft tumor volumes were reduced approximately 20% following mAb treatment. These data implicate the C-terminal of DCLK1 as a potential signal transduction regulator of DCLK1 in cancer via tumor suppressor microRNA related mechanism similar to siRNA mediated knockdown of DCLK1. These results suggest that utilizing mAb targeting DCLK1 may represent a potential novel therapeutic strategy to reduce the tumor stem-like cell subpopulation in PDAC.

Citation Format: Dongfeng Qu, Nathaniel Weygant, Randal May, Parthasarathy Chandrakesan, Daniel Owen, Sripathi Sureban, Courtney Houchen. DCLK1 targeted monoclonal antibodies demonstrate therapeutic potential against pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-48. doi:10.1158/1538-7445.AM2014-LB-48
Abstract LB-53: Regulatory role of miR-142-3p on the functional hepatic cancer stem cell marker CD133
Authors: Ng Kai Y. , Chai Stella , Tong Man , Kwan Pak Shing , Chan Yuen Piu , Lee Terence Kin Wah , Wong Nathalie , Guan Xin-Yuan , Ma Stephanie
Published: May 2015

Tumor relapse after therapy typifies hepatocellular carcinoma (HCC) and is believed to be attributable to residual cancer stem cells (CSCs) that survive initial treatment. We have previously identified a CSC population derived from HCC that is characterized by the expression of the transmembrane glycoprotein, CD133. Despite our growing knowledge of the importance of a functional CD133+ liver CSC subset in driving HCC, the regulatory mechanism of CD133 is not known. Epigenetic changes are believed to be essential in the control of cancer and stem cells. We report here the dynamic epigenetic regulation of the functional liver CSC marker CD133 by promoter methylation and miR-142-3p regulation. Unlike in other tumor types, we found DNA methylation to only play a minor role in the control of CD133 expression in HCC. More importantly, our results revealed that miR-142-3p plays an integral part in the direct targeting of CD133. The interaction between the 3’UTR of CD133 and miR-142-3p was identified by in silico prediction and substantiated by luciferase reporter analysis. Expression of CD133 was found to be inversely correlated with miR-142-3p in a panel of liver cell lines and HCC clinical samples. Functional studies with miR-142-3p stably transduced in HCC cells demonstrated a diminished ability to self-renew, initiate tumor growth, invade, migrate, induce capillary tube formation in endothelial cells and resist standard chemotherapy. Rescue experiments whereby CD133 and miR-142-3p is simultaneously overexpressed compensated the deregulated ability of the cells to confer these cancer and stem cell-like features. In summary, our findings suggestion promoter methylation to only play a minor role in the regulation of CD133 in HCC; and that miR-142-3p directly targets CD133 to regulate its ability to confer cancer and stem cell-like features in HCC.

Citation Format: Kai Yu Ng, Stella Chai, Man Tong, Pak Shing Kwan, Yuen Piu Chan, Terence Kin Wah Lee, Nathalie Wong, Xin-Yuan Guan, Stephanie Ma. Regulatory role of miR-142-3p on the functional hepatic cancer stem cell marker CD133. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-53. doi:10.1158/1538-7445.AM2014-LB-53
Abstract LB-58: Role of mitochondrial ROS in activation of glioma stem cells and promotion of cancer development
Authors: Wang Feng , Yuan Shuqiang , Chen Gang , Kim Sangbae , Yang Jing , Li Feng , Lee Ju-Seog , Li Xiao-Nan , Xu Rui-hua , Huang Peng
Published: May 2015

Glioblastoma stem cells (GSCs) play a major role in brain tumor development, and they can be induced to undergo apparent differentiation morphology by serum. It is generally assumed that induction of differentiation by serum in cancer stem cells would reduce their ability to form tumors. However, how serum triggers such change in GSC differentiation status and whether it really affects their ability to form tumor remain unclear. Here we show that serum exposure activates mitochondrial respiration and causes an increase in mitochondrial ROS and stress responses, leading to NFκB activation and down-regulation of stem cell markers including CD133, Olig2, SOX2 and Notch1. Chemical perturbation of mitochondrial electron transport chain causes ROS increase in serum-induced GSCs and further decreases stem cell markers, while antioxidants reduce ROS and prevent the loss of stem markers. Surprisingly, the serum-induced GSCs exhibit greater ability to form tumor in both orthotopic and subcutaneous xenograft models, which can be suppressed by antioxidants. Our study suggests that stimulation of mitochondrial respiration and ROS generation is critical in activation of GSCs and promoting tumor development in vivo. Although exposure of glioma stem cells to serum causes a decrease in stem cell markers and a loss of neurosphere morphology, the seemingly differentiated GSCs surprisingly exhibit greater ability to form tumors in vivo. A decrease in expression of certain stem cell markers such as CD133 and SOX2 may not necessarily indicate a decrease in tumorigenicity in vivo, and that redox signaling may play a major role in regulation of tumor formation from cancer stem cells.

Citation Format: Feng Wang, Shuqiang Yuan, Gang Chen, Sangbae Kim, Jing Yang, Feng Li, Ju-Seog Lee, Xiao-Nan Li, Rui-hua Xu, Peng Huang. Role of mitochondrial ROS in activation of glioma stem cells and promotion of cancer development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-58. doi:10.1158/1538-7445.AM2014-LB-58
Abstract LB-60: Targeting stem cells in triple negative breast cancer through combined MEK and AKT inhibition
Authors: Zhang Kevin , McDermott Sean P. , Wicha Max S.
Published: May 2015

Breast cancer stem cells (CSCs) are hypothesized to be involved in tumor metastasis, dormancy, and eventual recurrence. Among the molecular subtypes of breast cancer, triple negative (i.e. lacking HER2 amplification and estrogen and progesterone receptors) cancers have the highest frequency of CSCs with cytotoxic chemotherapy being the only established treatment option. Breast CSCs are relatively resistant to cytotoxic chemotherapy and radiation treatment indicating new treatment modalities are needed. Breast CSCs can be identified using flow cytometry with the cell surface marker profile CD44+CD24-EpCAM+ or by intracellular expression of aldehyde dehydrogenase (ALDH+). Previously, we identified a role for AKT signaling in the survival of breast CSCs. Here, we have tested if inhibition of MEK and AKT signaling with two small molecules, GSK1120212 and GSK690693, respectively, were able to reduce the frequency and overall number of CD44+CD24-EpCAM+ and/or ALDH+ CSCs in multiple triple-negative breast cancer cell lines. Our results suggest that either the CD44+CD24-EpCAM+ or ALDH+ fraction are sensitive to three days of combined inhibitor treatment. Within the MDA-MB-468 cell line, there is more phospho AKT (S473) and phospho MEK1/2 (S218/S222) signaling in both ALDH+ and CD44+CD24-EpCAM+ cells compared to non-stem cells. Furthermore, there is more phospho MEK1/2 in ALDH+ cells compared to CD44+CD24-EpCAM+ and more phospho AKT in CD44+CD24-EpCAM+ cells compared to ALDH+ CSCs. Together, these results highlight the possibility of using a combination of these agents in clinical trials to target triple-negative breast CSCs.

Citation Format: Kevin Zhang, Sean P. McDermott, Max S. Wicha. Targeting stem cells in triple negative breast cancer through combined MEK and AKT inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-60. doi:10.1158/1538-7445.AM2014-LB-60
Abstract LB-62: Inhibitor of differentiation 4 (ID4) maintains mammary stem cell homeostasis and identifies a poor-prognosis subset of basal-like breast cancers with a putative stem cell of origin
Authors: Junankar Simon , Baker Laura , Yun Kyuson , Swarbrick Alexander
Published: May 2015

Basal-like breast cancers (BLBC) are heterogenous, poorly differentiated and present with limited therapeutic opportunities and a poor prognosis. We have investigated the role of the HLH transcription factor ID4 in breast stem cells and basal breast cancer aetiology. Using an Id4GFP knock-in mouse and single cell gene expression studies we show that ID4 defines a subset of Cd24+Cd29hi basal mammary epithelial cells enriched for stem cell gene expression and mammary repopulating capacity. Homozygous deletion of Id4 followed by mammary transplantation reveals a cell-autonomous requirement for Id4 in mammary ductal morphogenesis in vivo. shRNA-mediated depletion of Id4 in the Comma-D mammary stem cell line in vitro shows that Id4 is required for epithelial cell proliferation. Conversely, over-expression of Id4 maintains the stem cell pool by restricting luminal differentiation of Comma-D cells. This effect of Id4 is mediated via suppression of BrCa1 expression and the Notch-Elf5 axis. In clinical breast cancer specimens, ID4 expression is absent in luminal tumours but very high in approximately half of all BLBCs, a proportion of which possess ID4 gene amplification. These tumours are characterized by unique transcriptomes with many features of mammary stem cells. Furthermore, ID4-positive BLBCs have a very poor prognosis across multiple independent tissue cohorts. This study identifies ID4 as a master regulator of mammary stem cell homeostasis and suggests that a subset of aggressive BLBCs derive from a primitive mammary stem or progenitor cell with basal characteristics.

Citation Format: Simon Junankar, Laura Baker, Kyuson Yun, Alexander Swarbrick. Inhibitor of differentiation 4 (ID4) maintains mammary stem cell homeostasis and identifies a poor-prognosis subset of basal-like breast cancers with a putative stem cell of origin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-62. doi:10.1158/1538-7445.AM2014-LB-62
Abstract LB-64: Immune modulatory effects of mesenchymal stem cells on regulatory T cells in mouse glioma model
Authors: Moon Kyung-Sub , Wainwright Derek A. , Balyasnikova Irina V. , Kim Chung Kwon , Ko Yoo Seung , Kim Kyung Keun , Lesniak Maciej S.
Published: May 2015

Objective: Although mesenchymal stem cells (MSCs) have been investigated as a vector in stem cell-based therapy for malignant glioma, there has been a debate about the exact role of MSCs in tumor microenvironment. From an immunnological aspect, MSCs have been reported to inhibit T cell proliferation and to increase CD4+FoxP3+ regulatory T cells (Tregs). They also have been investigated as a potent immune suppressor in several clinical situations including autoimmune diseases. However, whether MSCs have an immune modulatory effect on Tregs in malignant gliomas or not has not been established.

Materials & methods: The author explored the impact of MSCs on freshly sorted CD4+ T cells or already activated Tregs in vitro to determine whether MSCs have different influences on naive T cells and Tregs. Furthermore, using orthotropic mouse glioma model, the author investigated MSC's effect on Tregs during tumor progression.

Results: The authors observed that MSCs decreased Treg conversion from naive T cells and Treg proliferation, without affecting the proportion of natural / induced Treg [nTreg; CD4+FoxP3+Helios+ / iTreg; CD4+FoxP3+Helios-]. When co-cultured with already activated Tregs, however, MSCs did not affect Treg induction and proliferation, but decreased the proportion of iTreg. With an intracranially implanted GL261 cell-based orthotropic mouse model, co-injection of MSCs with tumor cell implantation slightly increased Treg recruit into the tumor and decreased the proportion of iTreg. When MSCs were injected after glioma formation, there was no significant effect of MSCs on Treg recruit and phenotypic changes. Furthermore, there was a lower level of cytokine expressions including TGF-β, INF-γ, IL-2, and IL-1β in Tregs cocultured with MSCs than that in solely cultured Tregs. Increased gene expression of IL-6, CCL17, and TGF-β, or decreased gene expression of CCL2 in MSCs may be relevant to the phenotypic changes of cocultured Tregs.

Conclusions: This study demonstrates that MSCs mainly inhibit iTreg, leading to a relative increase of the proportion of nTregs in mouse glioma model, possibly throughIL-6 signaling pathway. With a better understanding for critical factors and immunological consequences for the altered Treg phenotype in glioma, future experiments will be needed to elucidate the immunological impact of MSCs on Tregs in malignant glioma.

Citation Format: Kyung-Sub Moon, Derek A. Wainwright, Irina V. Balyasnikova, Chung Kwon Kim, Yoo Seung Ko, Kyung Keun Kim, Maciej S. Lesniak. Immune modulatory effects of mesenchymal stem cells on regulatory T cells in mouse glioma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-64. doi:10.1158/1538-7445.AM2014-LB-64
Abstract LB-7: Design and development of a novel series of orally active, selective PI3Kp110β/δ inhibitors for the treatment of cancer
Authors: Shuttleworth Stephen J.
Published: May 2015

The phosphatidylinositide 3-kinases (PI3Ks) have emerged as attractive therapeutic targets for the treatment of cancer. The majority of PI3K inhibitors currently in clinical development primarily target either all four class I enzymes (p110α, β, δ and γ) with limited selectivity, or are pan-class I/mTOR dual inhibitors. Over recent years, a switch of focus to more selective PI3K inhibitors for the treatment of specific tumor types has occurred. Of particular note, p110b has been reported to be the principal isoform in driving PTEN-deficient tumorigenesis; additionally, high levels of p110δ expression in solid tumor cells have been shown to suppress PTEN activity, generating cellular sensitivity to p110δ inhibition through PTEN activation. Moreover, PI3K pathway activation in certain PTEN-null solid tumors has been shown to be mediated by both the p110β and p110δ catalytic subunits, suggesting the potential of a dual-selective p110β/δ inhibitor in this setting. During the past 12 months, the discovery of activating mutant forms of both p110β and p110δ has been reported. The strategy outlined in this presentation centres on the design and development of a new class of orally-active small molecules that selectively inhibit p110β and p110δ, and which display high degrees of specificity over protein kinases and other lipid kinases. Moreover, a spectrum of p110β/δ selectivity has been achieved in this series, which has facilitated interrogation of preferred isoform inhibition profiles required to confer tumor growth inhibition; additionally, by tuning selectivity over the other class I isoforms, elimination of the mechanism-related toxicities associated with the pan-class I inhibitors currently in the clinic can be realized, making such agents potentially more conducive to combination therapy. In vitro and in vivo therapy data for advanced lead compounds will be disclosed.

Citation Format: Stephen J. Shuttleworth. Design and development of a novel series of orally active, selective PI3Kp110β/δ inhibitors for the treatment of cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-7. doi:10.1158/1538-7445.AM2014-LB-7
Abstract LB-74: The NLR-related protein NWD1 is associated with prostate cancer and modulates androgen receptor signaling
Authors: Correa Ricardo G. , Krajewska Maryla , Ware Carl F. , Gerlic Motti , Reed John C.
Published: May 2015

Prostate cancer (PCa) is among the leading causes of cancer-related death in men. Androgen receptor (AR) signaling plays a seminal role in prostate development and homeostasis, and dysregulation of this pathway is intimately linked to prostate cancer pathogenesis and progression. Here, we identify the cytosolic NLR-related protein NWD1 as a novel modulator of AR signaling. We determined that expression of NWD1 becomes elevated during prostate cancer progression, based on analysis of primary tumor specimens. Experiments with cultured cells showed that NWD1 expression is up-regulated by the sex-determining region Y (SRY) proteins. Gene silencing by short hairpin RNA (shRNA), in conjunction with transcriptional profiling, showed that NWD1 is required for expression of PDEF (prostate-derived Ets factor), which is known to bind and co-regulate AR. Of note, NWD1 modulates AR protein levels. Silencing NWD1 in PCa cell lines reduces the levels of androgen-stimulated accumulation of nuclear AR and suppresses activity of androgen-driven reporter genes. NWD1 knockdown potently suppressed growth of androgen-dependent LNCaP prostate cancer cells, thus showing its functional importance in an AR-dependent tumor cell model. Proteomics analysis suggested that NWD1 associates with various molecular chaperones commonly related to AR complexes. Altogether, our data suggest a role for tumor-associated over-expression of NWD1 in the dysregulation of AR signaling in PCa.

Citation Format: Ricardo G. Correa, Maryla Krajewska, Carl F. Ware, Motti Gerlic, John C. Reed. The NLR-related protein NWD1 is associated with prostate cancer and modulates androgen receptor signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-74. doi:10.1158/1538-7445.AM2014-LB-74
Abstract LB-75: Oct7 is expressed in human gliomas and correlates with malignancy grade
Authors: Rahman Mohummad Aminur , Leiss Lina , Saed Halala S. , Gjerde Christiane H. , Lellahi Mohammad S. , Mutlu Ercan , Enger Per Øyvind
Published: May 2015

INTRODUCTION: The transcription factor Oct 7 (also called POU3f2/Brn2) is expressed during neurogenesis, and constitutes the CNS equivalent of Oct 4, a critical regulator of induced pluripotent stem cells. Moreover, data suggest its expression is regulated by hypoxia, and Oct 7 expression has been reported in malignant melanoma of the skin. Since both melanomas and CNS-malignancies arise in organs of neuroectodermal origin, we investigated whether human gliomas expressed Oct7.

MATERIAL & METHODS: We performed immunohistochemistry of 150 grade II-IV gliomas from our tumor bank, and subsequently performed western blots of 20 glioma samples. In addition, we performed flow cytometry analysis of Oct 7 expression in 5 acutely dissociated tumors. Using lentiviral transfection we established Oct7 overexpression in a panel of constitutively negative glioma cell lines, as well as knock-down in an Oct7 positive glioma cell line, to investigate the effect of Oct 7 expression on proliferation, migration and differentiation.

RESULTS: Immunohistochemistry showed that Oct7 was almost uniformly expressed in human gliomas, although at a varying degree. Microscopy revealed a predominantly nuclear staining pattern, but cytoplasmic immunopositivity for Oct7 could also be detected in some tumors. Both western blot and flow cytometry confirmed expression of Oct7 in human gliomas. With two independent observers we obtained a nuclear staining index for all tumors, with 61% positive nuclei in GBM specimens, which was significantly higher than for grade II (37%) and grade III (36%) tumors (p= 0.0001). Moreover, ongoing studies suggest that overexpression of Oct7 increases the proportion of cells in G2/M phase of the cell cycle, suggesting that this transcription factor has a role in regulating tumor cell proliferation, and hence possibly overall tumor aggressiveness. Ongoing studies aim at further elucidating the multiple roles of Oct7 in brain tumor progression.

Citation Format: Mohummad Aminur Rahman, Lina Leiss, Halala S. Saed, Christiane H. Gjerde, Mohammad S. Lellahi, Ercan Mutlu, Per Øyvind Enger. Oct7 is expressed in human gliomas and correlates with malignancy grade. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-75. doi:10.1158/1538-7445.AM2014-LB-75
Abstract LB-76: Absolute single-molecule sensitive NGS by SPLITSEQ: a new ctDNA analysis tool
Authors: Lebofsky Ronald , Madic Jordan , Saliou Adrien , Bidard Francois-Clement , Lantz Olivier , Stern Marc-Henri , Pierga Jean-Yves
Published: May 2015

Circulating tumor DNA (ctDNA) analysis requires absolute single molecule sensitivity, since only a few copies of the mutation may be present per blood draw. Recent developments in next-generation sequencing (NGS) sample preparation have improved the relative sensitivity to detect <0.01% variants in background. However, when the total number of input mutant molecules falls below 10, these improvements have not translated into the absolute sensitivity required to sequence these rare templates. Here we develop and present SPLITSEQ, a novel microfluidics and NGS-based method that combines 0.01% fractional sensitivity with absolute single copy input sensitivity in an excess of background DNA. We provide multiplexed performance data on mutations in KRAS codons 12 and 13 and BRAF V600E, targets that are currently being developed as circulating predictive and prognostic biomarkers in colorectal and other cancers. Using reconstituted DNA solutions from mutant and normal cell lines ranging from 0.01% to 10% mutated allele fractions, we detected the sequence of approximately 0.2, 2, and 20 nominal mutated KRAS and BRAF inputs total in 10 000X background. Single copy numbers were supported by droplet digital PCR (ddPCR) data, an orthogonal single-molecule sensitive technique. Lastly, we confirm the specificity of the technique by screening wild-type DNA only and finding a false positive rate significantly lower than ddPCR. Moreover, in comparison with digital PCR, SPLITSEQ is easily amenable to high levels of multiplexing and detects non-recurrent mutations in tumor suppressors along the length of the sequence. Since the only requirement is a single PCR-amplifiable template, SPLITSEQ fills an unmet need in ctDNA detection, providing biomarkers from hereto unanalyzable liquid biopsies containing rare mutants.

Citation Format: Ronald Lebofsky, Jordan Madic, Adrien Saliou, Francois-Clement Bidard, Olivier Lantz, Marc-Henri Stern, Jean-Yves Pierga. Absolute single-molecule sensitive NGS by SPLITSEQ: a new ctDNA analysis tool. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-76. doi:10.1158/1538-7445.AM2014-LB-76
Abstract LB-77: The Met receptor tyrosine kinase drives signaling through EGFR and ErbB3 in Met-amplified non-small-cell lung cancer
Authors: Stern Yaakov E. , Lai Andrea Z. , Park Morag
Published: May 2015

Receptor tyrosine kinases are canonically activated by dimerization induced by the binding of a cognate ligand. This leads to autophosphorylation of the receptor molecules by their intrinsic kinase domains and the recruitment of intracellular protein complexes to initiate signaling. In many human cancers, receptor tyrosine kinases are aberrantly activated to promote proliferative signaling, thus driving tumor growth. The MET proto-oncogene is amplified in approximately 5% of non-small-cell lung cancers (NSCLC), a disease in which aberrant signaling through the epidermal growth factor receptor (EGFR) or the anaplastic lymphoma kinase is known to be oncogenic. The Met receptor tyrosine kinase is overexpressed and constitutively active in cell lines derived from MET-amplified tumors, and Met activity is required for proliferation. Furthermore, Met activity is sufficient to promote the phosphorylation of EGFR and its homologs ErbB2 and ErbB3 in the presence of inhibitors targeting EGFR, ErbB2, and Src family kinases. We show that Met utilizes the EGFR, ErbB2 and ErbB3 proteins as scaffolds to broaden downstream oncogenic signaling and promote cancer cell proliferation. The contribution of EGFR and its homologs to Met signaling in NSCLC will be presented with a focus on ErbB family kinases required for proliferation in MET-amplified NSCLC. Understanding the mechanisms by which Met drives proliferative signaling in NSCLC will become crucial as Met-targeted therapies become available in the clinic.

Citation Format: Yaakov E. Stern, Andrea Z. Lai, Morag Park. The Met receptor tyrosine kinase drives signaling through EGFR and ErbB3 in Met-amplified non-small-cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-77. doi:10.1158/1538-7445.AM2014-LB-77
Abstract LB-78: Effects of rapamycin and fasting-cycles therapy on differential stress resistance and sensitization in breast cancer mouse models
Authors: Biase Stefano D. , Brandorst Sebastian , Wei Min , Longo Valter
Published: May 2015

Short-term starvation (STS) has been shown to be effective in both protection of normal cell and tissues and the sensitization of a variety of tumors during chemotherapy treatment. This condition of protection/sensitization of different cell types under the same condition has been named differential stress resistance (DSR). Although the effects of fasting have been mainly attributed to reduced glucose and circulating IGF1 levels, the involvement of signaling genes regulated by glucose and growth factors in these effects of fasting remain poorly understood.

In this study we have administered the mTor inhibitor rapamycin in combination with STS and with the chemotherapy drug doxorubicin to shed light on the role of mTOR in the STS response and on its role in mediating DSR.

In a mouse allograft model for breast cancer (4T1) we show that STS and rapamycin have an additive effect in reducing tumor progression, which confirms the possibility that STS-mediated effects on cancer cells are not due to the up-regulation of mTOR. Surprisingly, we observed that the administration of rapamycin during chemotherapy sensitizes the mice to the drug leading to an increased mortality. However, this sensitizing effect could be reversed by STS.

In summary, the combination of mTor inhibition by rapamycin and STS have additive effects in retarding breast cancer tumor growth and also prevent the chemotherapy-dependent sensitization of normal cells caused by rapamycin alone. Because rapamycin is used for the treatment of certain tumors in humans, these results may have important implications for the safety of therapies using a combination of rapamycin and chemotherapy and indicate that the combination of Tor inhibitors and STS should be tested clinically.

Citation Format: Stefano Di Biase, Sebastian Brandorst, Min Wei, Valter Longo. Effects of rapamycin and fasting-cycles therapy on differential stress resistance and sensitization in breast cancer mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-78. doi:10.1158/1538-7445.AM2014-LB-78
Abstract LB-80: Exome sequencing of murine p53-knockout T-cell lymphomas reveals Pten loss and Cdk6 amplification as key steps toward cellular transformation
Authors: Dudgeon Crissy , Chan Chang , Sun Yvonne , Levine Arnold J.
Published: May 2015

Loss of the tumor suppressor, p53, occurs in over 50% of cancers. The ability for p53 to protect the cell from tumorigenesis is based on its ability to induce cell cycle arrest, apoptosis, and senescence following genotoxic stress. Furthermore, genetic deletion of p53 in mice gives rise to spontaneous thymic (T-cell) lymphomas and sarcomas. In this study, we used the p53-knockout (KO) mouse as a model for progression of tumorigenesis. We first analyzed genomic DNA of p53-KO and WT normal thymus at various ages and p53-KO thymic lymphomas using TCRβ sequencing, allowing us to determine the clonality of the T cell repertoire. These data suggest that p53-KO thymic lymphomas were mostly oligoclonal, with some tumors comprising of a dominant clone. We further analyzed tumors with dominant clones for genomic instability by exome sequencing. Sequencing analysis revealed that there were many non-synonymous mutations occurring in each tumor, but with no similarities between tumors. On the other hand, all of the murine tumors had deletion of Pten and amplification of Cdk6. This is in stark contrast to what is seen in most human T cell lymphoblastic leukemias, which have activating mutations in Notch1 and loss of Arf. Interestingly, loss of Pten not only occurred in all tumors, but also in most, if not all, clones in the tumor. This suggests that Pten loss is an early event driving T cell transformation in a p53-KO background. Using this data, we found the somatic mutation rate in the tumors to be 1000-fold greater than the germline mutation rate. Loss of p53 also caused chromosomal abnormalities as seen with increased copy number variations. Taken together, our work suggests that, with initial loss of p53, the path to T cell tumorigenesis is vastly different from that of human tumors. Without p53, the somatic genome increases in mutation rate and copy number variations, thus solidifying p53's role as guardian of the genome.

Citation Format: Crissy Dudgeon, Chang Chan, Yvonne Sun, Arnold J. Levine. Exome sequencing of murine p53-knockout T-cell lymphomas reveals Pten loss and Cdk6 amplification as key steps toward cellular transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-80. doi:10.1158/1538-7445.AM2014-LB-80
Abstract LB-81: Conditional deletion of the tumor suppressor Hic1 results in aneuploidy and single-step transformation
Authors: Szczepny Anette , McKenzie Lisa , Jayesekara Samantha , Russell Prue , Wright Gavin , Baylin Stephen B. , Rossello Fernando J. , Cain Jason E. , Watkins David N.
Published: May 2015

HIC1 is a zinc-finger transcriptional repressor frequently targeted for deletion and/or epigenetic gene silencing in cancer. In cancer cell lines, re-expression of HIC1 results in the downregulation of target genes thought to promote context-dependent tumor growth, including Sirt1, Atoh1, Efna1 and components of the WNT and STAT3 signalling pathway.

In mouse embryonic fibroblasts, we now show that deletion of a conditional loxP allele of Hic1 results in immediate growth arrest without significant overexpression of Hic1 target genes, followed by a prolonged period of cellular quiescence. After several weeks, we then observed the spontaneous emergence of rapidly growing clones that fulfil all the criteria for transformation, associated with aneuploidy and recurrent copy number alterations. By contrast, conditional deletion of p53 using the same experimental protocol resulted in prompt immortalisation without transformation, and a stable tetraploid karyotype resembling that seen in NIH-3T3 cells. These data suggest that inactivation of Hic1 in cells undergoing replication stress in tissue culture can induce complete transformation associated with marked genomic instability.

To test this hypothesis in vivo, we employed the inhaled adenoviral Cre recombinase model in order to direct loxP recombination to the airway epithelium of adult mice. As previously described by other groups, Cre activation of a conditional oncogenic KRasG12D mutant resulted in the appearance of multiple lung adenomas within 6-8 weeks following viral inhalation. By contrast, mice carrying this KRasG12D allele, as well as homozygous for the conditional Hic1lox knockout allele, developed highly aggressive adenocarcinomas with prominent pleomorphic and micropapillary histology. These data suggest a novel tumour suppressor role for HIC1 in maintaining genomic stability in replicating cells. HIC1 is known to interact with components of the SWI/SNF and PRC2 complexes, which help maintain genomic integrity by re-establishing constitutive heterochromatin following DNA replication. We therefore speculate that loss of HIC1 function may phenocopy recently described mutations in components of these chromatin-remodelling complexes such as ARID1A and BRG1.

Citation Format: Anette Szczepny, Lisa McKenzie, Samantha Jayesekara, Prue Russell, Gavin Wright, Stephen B. Baylin, Fernando J. Rossello, Jason E. Cain, David N. Watkins. Conditional deletion of the tumor suppressor Hic1 results in aneuploidy and single-step transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-81. doi:10.1158/1538-7445.AM2014-LB-81