bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3011: Dietary fat, fatty acids, and ovarian cancer risk: Preliminary findings from the Shanghai Women’s Health Study
Authors: Akam Eftitan Y. , Murff Harvey J. , Xiang Yong-Bing , Khankari Nikhil K. , Cai Hui , Shu Xiao O. , Zheng Wei , Beeghly-Fadiel Alicia
Published: July 2017

Introduction: Dietary fat is an essential nutrient for human growth and development, but epidemiological studies have linked excess fat to the development of multiple malignancies. The few existing studies on ovarian cancer risk are inconsistent, possibly due to differences in sources and subtypes of fat. For example, omega-3 (n-3) polyunsaturated fatty acids (PUFAs) are thought to be antineoplastic, while omega-6 (n-6) PUFAs may promote carcinogenesis.

Approach: We evaluated dietary fat in relation to ovarian cancer risk in the prospective Shanghai Women’s Health Study (SWHS). Intake levels were determined from food frequency questionnaires (FFQ) administered at baseline and during follow-up; specific nutrient components were based on the sum of specific foods multiplied by nutrient content from the Chinese Food Composition Table. Cox proportional hazards regression was used to estimate relative risks (RR) and 95% confidence intervals (CI) for dietary fat and fatty acid components categorized into quintiles. Minimal models included age and residual adjustment for total energy intake; fully adjusted models also included education, years of menstruation, years of oral contraceptive use, parity, years of breastfeeding, family history of ovarian cancer, smoking, drinking, NSAID use, BMI, and physical activity.

Results: Among 73,144 SWHS participants, we identified a total of 219 ovarian cancer cases in 1,114,828 person-years of follow-up. We found significant dose-response relationships between increasing total PUFA (P-trend=0.032) and n-6 PUFA (P-trend=0.022) intakes and decreased ovarian cancer risk. In both minimally and fully adjusted models, women with the highest quintile of total dietary PUFAs were approximately 35% less likely to develop ovarian cancer (RR=0.65, 95% CI: 0.43-1.00), and women with the highest n-6 PUFA intake had a nearly 40% lower risk (RR=0.61, 95% CI: 0.40-0.93). Total fat, monounsaturated fat, and n-3 PUFA intake were not significantly related to ovarian cancer risk.

Conclusions: Findings from our preliminary analysis of a large prospective cohort of Chinese women suggest that total PUFA intake may be protective of ovarian cancer, and that this association may be driven by n-6 rather than n-3 PUFAs. Further evaluation, including stratification, sensitivity analysis, and evaluation of source of dietary fatty acids, is currently underway.

Citation Format: Eftitan Y. Akam, Harvey J. Murff, Yong-Bing Xiang, Nikhil K. Khankari, Hui Cai, Xiao O. Shu, Wei Zheng, Alicia Beeghly-Fadiel. Dietary fat, fatty acids, and ovarian cancer risk: Preliminary findings from the Shanghai Women’s Health Study [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 3011. doi:10.1158/1538-7445.AM2017-3011
Abstract 302: A novel epigenetic pathway in medulloblastoma
Authors: Ayad Nagi , Penas Clara
Published: July 2017

Medulloblastoma is the most common malignant pediatric brain tumor with variable prognosis due to its clinical and genomic heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from this disease. Therefore there is a need to develop novel therapies for patients. We recently reported that Casein kinase 1δ (CK1δ) may be an attractive therapeutic target for medulloblastoma. CK1δ is a serine/threonine kinase that controls cell cycle progression, signal transduction and neurogenesis. We found high levels of CK1δ protein in mouse models of medulloblastoma and human medulloblastoma samples. Furthermore, CK1δ inhibition dramatically reduced medulloblastoma tumor progression. We demonstrate here that CK1δ phosphorylates the epigenetic reader bromodomain-containing protein 4 (BRD4). BRD4 has been identified as a therapeutic target in several cancers, including medulloblastoma. We demonstrate that CK1δ phosphorylates BRD4 and that CK1δ is required for BRD4 binding to the Gli1 promoter in vitro and in vivo. Furthermore, combined CK1δ/BRD4 inhibition is a novel means of reducing medulloblastoma growth downstream of SUFU and SMO. These studies define a novel therapeutic means of inhibiting SMO inhibitor resistant medulloblastoma.

Citation Format: Nagi Ayad, Clara Penas. A novel epigenetic pathway in medulloblastoma [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 302. doi:10.1158/1538-7445.AM2017-302
Abstract 3022: Novel hydrocarbon stapled peptide inhibitors suppresses β-catenin dependent cancer growth and immunomodulates colorectal tumors
Authors: Zhu David , Jin Joy
Published: July 2017

Mutations in several components of the canonical Wnt pathway function as drivers in a range of common solid tumor cancers, including about 90% of sporadic colorectal cancers (CRCs), the third most common cancer in the US. In particular, activation of β-catenin (β-cat), a key Wnt signaling component, can initiate tumorigenesis in the intestine and promote cancer stem cell metastasis6,7. As such, targeting the Wnt pathway is an attractive approach to the development of cancer therapeutics. However, Wnt inhibitors tested to date, including those that disrupt β-cat activity, have either had limited efficacy or unacceptable side effect profiles1. Here, we developed a potent and robust, selective lead compound targeting oncogenic Wnt activity by disrupting the highly specific interaction of β-cat with its co-activators BCL9/B9L, which has been shown to abrogate transactivation. By using hydrocarbon stapled peptide technology and an acutely sensitive high throughput biochemical screening assay, we have identified several promising peptides that possess a strong pharmacokinetic profile. These peptides (i) exhibit robust activity in mouse models; (ii) inhibit cancer cell proliferation, metastasis, and stem cell progression; and (iii) modulate the immune microenvironment, demonstrating the feasibility of such peptide development and selection. Synergy is demonstrated between the stapled peptides and anti-PD1 antibodies, which has significant implications for combination therapy with commercial immune checkpoint blockers that have become standard of care for second-line melanoma treatment. These peptides represent a promising new potential therapeutic class for patients with CRC, for which traditional chemotherapy and targeted inhibitors have had limited therapeutic success.

Citation Format: David Zhu, Joy Jin. Novel hydrocarbon stapled peptide inhibitors suppresses β-catenin dependent cancer growth and immunomodulates colorectal 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 3022. doi:10.1158/1538-7445.AM2017-3022
Abstract 3028: Reactive oxygen species induced nuclear translocation of c-MET via INTERNET mechanism
Authors: Chen Mei-Kuang , Du Yi , Chu Yu-Yi , Hung Mien-Chie
Published: July 2017

c-MET is one of the receptor tyrosine kinases (RTKs) that are overexpressed in malignant cancer types including breast cancer. c-MET is traditionally known for its role in signaling transduction from the cell surface, our recent publication provided evidence that c-MET can be translocated into the nucleus and phosphorylate PARP1 in response to reactive oxygen species (ROS) stimulation (Du et al. Nat Med 2016). Oxidative stress is a common phenomenon in cancer cells due to alteration of metabolism, and constitutive oxidative stress related to ROS has been observed in breast cancer cells. In this study, we demonstrated that hepatocyte growth factor (HGF) as well as hydrogen peroxide (H2O2) can induce retrograde translocation of c-MET holoreceptor via a membrane-bound vesicle transport mechanism and translocate into the nucleus through INTERNET (integral trafficking from the ER to the nuclear envelope transport pathway) (Wang et al. J Biol Chem 2012) in breast cancer cells. While c-MET nuclear transportation increases dramatically under H2O2 treatment, our findings provide a putative mechanism by which breast cancer cells adapt to oxidative stress and suggest that INTERNET might be a general nuclear translocation mechanism shared among RTKs.

Citation Format: Mei-Kuang Chen, Yi Du, Yu-Yi Chu, Mien-Chie Hung. Reactive oxygen species induced nuclear translocation of c-MET via INTERNET mechanism [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 3028. doi:10.1158/1538-7445.AM2017-3028
Abstract 3044: Rapid decay of engulfed extracellular miRNA by XRN1 exonuclease promotes transient epithelial-mesenchymal transition
Authors: Zangari Joséphine , Ilie Marius , Rouaud Florian , Signetti Laurie , Ohanna Mickaël , Didier Robin , Roméo Barnabé , Goldoni Dana , Nottet Nicolas , Staedel Cathy , Gal Jocelyn , Mari Bernard , Mograbi Baharia , Hofman Paul , Brest Patrick
Published: July 2017

Background: Extracellular vesicles (EVs) have been shown to play an important role in intercellular communication as carriers of DNA, RNA and proteins. While the intercellular transfer of miRNA thorough EVs has been widely studied, the stability of extracellular miRNA (ex-miRNA) once engulfed by recipient cell remains to be determined.

Materials and Methods: Lung cancer cells (A549 cell line) were incubated with neutrophils or their supernatant or isolated EVs from these supernatant. Kinetics of expression of miR-223-3p were followed by RT-qPCR. FOXO1 expression and EMT expression markers were used as endpoints to follow effective effect or miR-223-3p in recipient cells.

Results: We identified the ex-miRNA-directed phenotype to be transient due to the rapid decay of the ex-miRNA. We demonstrated that the ex-miR-223-3p transferred from neutrophils to cancer cells was functional as demonstrated by the decreased expression of its target, FOXO1, and the occurrence of epithelial-mesenchymal transition reprogramming. We showed that the engulfed ex-miRNA, unlike endogenous miRNA, was unstable, enabling dynamic regulation and a return to a non-invasive phenotype within less than 8 hours. This transient phenotype could be modulated by targeting XRN1/PACMAN exonuclease. Indeed, its silencing was associated with slower decay of ex-miR-223-3p and subsequently prolonged the invasive properties.

Conclusion: We showed that the “steady step” level of engulfed miRNA (ex-miR-223-3p) and its subsequent activity was dependent on the presence of a donor cell in the surroundings to constantly fuel the recipient cell with ex-miRNAs and of XRN1 exonuclease, which is involved in the decay of these imported miRNAs.

Citation Format: Joséphine Zangari, Marius Ilie, Florian Rouaud, Laurie Signetti, Mickaël Ohanna, Robin Didier, Barnabé Roméo, Dana Goldoni, Nicolas Nottet, Cathy Staedel, Jocelyn Gal, Bernard Mari, Baharia Mograbi, Paul Hofman, Patrick Brest. Rapid decay of engulfed extracellular miRNA by XRN1 exonuclease promotes transient epithelial-mesenchymal transition [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 3044. doi:10.1158/1538-7445.AM2017-3044
Abstract 3048: A noncoding function of TYRP1 mRNA promotes melanoma growth
Authors: Galibert Marie-Dominique , Gilot David , Migault Mélodie , Bachelot Laura , Journé Fabrice , Rogiers Aljosja , Donnou-Fournet Emmanuelle , Mogha Ariane , Mouchet Nicolas , Pinel-Marie Marie-Laure , Mari Bernard , Montier Tristan , Corre Sébastien , Gautron Arthur , Rambow Florian , Hajj Petra El , Jouira Rania Ben , Tartare-Deckert Sophie , Marine Jean-Christophe , Felden Brice , Ghanem Ghanem
Published: July 2017

RNAs competition to bind miRNA has been proposed to influence biological systems whether these RNAs are implicated in diseases onset is unclear. Here, we report that TYRP1 mRNA, in addition to encoding tyrosinase-related protein 1 (TYRP1), indirectly governs cell proliferation by sequestering miR-16 on non-canonical miRNA response elements (MREs). Consequently, sequestered miR-16 is no longer able to repress its mRNA targets such as RAB17, which we show is involved in melanoma cell proliferation and invasion. Restoration of miR-16 tumor suppressor function can be achieved in vitro by silencing TYRP1 or increasing miR-16 expression levels. Importantly, TYRP1-dependent miR-16 sequestration can also be overcome in vivo using small oligonucleotides that mask miR-16 binding sites on TYRP1 mRNA. Together, our findings assign a pathogenic noncoding function to TYRP1 mRNA and underscore miRNA displacement as a new antimelanoma targeted therapeutic approach.

Citation Format: Marie-Dominique Galibert, David Gilot, Mélodie Migault, Laura Bachelot, Fabrice Journé, Aljosja Rogiers, Emmanuelle Donnou-Fournet, Ariane Mogha, Nicolas Mouchet, Marie-Laure Pinel-Marie, Bernard Mari, Tristan Montier, Sébastien Corre, Arthur Gautron, Florian Rambow, Petra El Hajj, Rania Ben Jouira, Sophie Tartare-Deckert, Jean-Christophe Marine, Brice Felden, Ghanem Ghanem. A noncoding function of TYRP1 mRNA promotes melanoma growth [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 3048. doi:10.1158/1538-7445.AM2017-3048
Abstract 3049: Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing
Authors: Demeulemeester Jonas , Kumar Parveen , Møller Elen K. , Nord Silje , Wedge David C. , Peterson April , Mathiesen Randi R. , Fjelldal Renathe , Esteki Masoud Z. , Theunis Koen , Gallardo Elia F. , Grundstad Jason , Borgen Elin , Baumbusch Lars O. , Børresen-Dale Anne-Lise , White Kevin P. , Kristensen Vessela N. , Loo Peter Van , Voet Thierry , Naume Bjørn
Published: July 2017

Background. Single-cell micro-metastases of solid tumors often occur in the bone marrow. These disseminated tumor cells (DTCs) may resist therapy and lay dormant or progress to cause overt bone and visceral metastases. The molecular nature of DTCs remains elusive, as well as when and from where in the tumor they originate. Here, we apply single-cell sequencing to identify and trace the origin of DTCs in breast cancer.

Results. We sequence the genomes of 63 single cells isolated from six non-metastatic breast cancer patients. By comparing the cells’ DNA copy number aberration (CNA) landscapes with those of the primary tumors and lymph node metastasis, we establish that 53% of the single cells morphologically classified as tumor cells are DTCs disseminating from the observed tumor. The remaining cells represent either non-aberrant ‘normal’ cells or ‘aberrant cells of unknown origin’ that have CNA landscapes discordant from the tumor. Further analyses suggest that the prevalence of aberrant cells of unknown origin is age-dependent, and that at least a subset is hematopoietic in origin. Evolutionary reconstruction analysis of bulk tumor and DTC genomes enables ordering of CNA events in molecular pseudo-time and traced the origin of the DTCs to either the main tumor clone, primary tumor subclones, or subclones in an axillary lymph node metastasis.

Conclusions. Single-cell sequencing of bone marrow epithelial-like cells, in parallel with intra-tumor genetic heterogeneity profiling from bulk DNA, is a powerful approach to identify and study DTCs, yielding insight into metastatic processes. A heterogeneous population of CNA-positive cells is present in the bone marrow of non-metastatic breast cancer patients, only part of which are derived from the observed tumor lineages.

Citation Format: Jonas Demeulemeester, Parveen Kumar, Elen K. Møller, Silje Nord, David C. Wedge, April Peterson, Randi R. Mathiesen, Renathe Fjelldal, Masoud Z. Esteki, Koen Theunis, Elia F. Gallardo, Jason Grundstad, Elin Borgen, Lars O. Baumbusch, Anne-Lise Børresen-Dale, Kevin P. White, Vessela N. Kristensen, Peter Van Loo, Thierry Voet, Bjørn Naume. Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing [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 3049. doi:10.1158/1538-7445.AM2017-3049
Abstract 3054: Tracing EMT with fluorescent biosensors (chromobodies) in living cancer cells
Authors: Maier Julia , Plaga Theresa , Egetemaier Stefanie , Traenkle Bjoern , Rothbauer Ulrich
Published: July 2017

Epithelial-mesenchymal transition (EMT) is a complex cellular program, involved in the progression of epithelial cancers to a metastatic stage. During this process characteristic epithelial marker proteins are repressed in favor of mesenchymal biomarkers, such as the intermediate filament protein vimentin. There is an ongoing need for reliable cell-based EMT model systems, which can be adapted to screening campaigns for novel anti-metastatic therapies. Imaging-based screening systems are restricted to endpoint immunofluorescence studies that provide only little information about the dynamics of the process, or to ectopic expression of EMT markers, fused to fluorescent proteins. However, overexpression of EMT marker proteins, particularly vimentin, have been shown to influence the EMT phenotype of the cells.

Here we introduce chromobodies as an innovative technology to monitor EMT in living non-small lung cancer cells. Chromobodies are fluorescently-labeled single-domain-antibodies that are functional within in living cells and directly target endogenous proteins. We generated chromobodies against the EMT biomarkers vimentin, actin and β-catenin and stably introduced them into the genome of A549 cells. Upon induction of EMT with transforming growth factor β (TGF- β), we could visualize the dynamic reorganization of endogenous vimentin, actin and β-catenin in the established EMT chromobody models in 2D as well as 3D cell culture. By means of these chromobody cell lines we further developed a 2D high content analysis system to visualize and quantify spatiotemporal changes of the endogenous biomarkers vimentin and actin during the EMT process. In a proof-of-principle screen, using known EMT-affecting reagents (e.g. TGF receptor I inhibitors LY364947 and SB505124) as well as EMT-unrelated agents, we were able to show that this high content analysis system can be applied to select compounds that inhibit TGF-β-induced EMT.

In summary, live cell imaging of EMT markers with chromobodies provides a novel versatile approach to study EMT in real time and allows the identification of EMT inhibitors as potential candidates for anti-metastatic therapies by high content screening.

Citation Format: Julia Maier, Theresa Plaga, Stefanie Egetemaier, Bjoern Traenkle, Ulrich Rothbauer. Tracing EMT with fluorescent biosensors (chromobodies) in living cancer cells [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 3054. doi:10.1158/1538-7445.AM2017-3054
Abstract 3059: UHRF1 deficiency exerts anti-tumor immune responses in non-small cell lung cancer
Authors: Xiang Handan , Chen Junying , Wang Xiao-Fan
Published: July 2017

Immune surveillance is essential for effective tumor clearance. However, the tumor microenvironment is typically impermissive for proper stimulation of both innate and adaptive immune populations. Epigenetic inhibitors have recently been shown to function as immune sensitizing reagents in the suppressive microenvironment. In our search for novel epigenetic regulators with immunomodulatory functions, we found ubiquitin-like with PHD and ring finger domain-containing protein 1 (UHRF1) being crucial for the suppression of immune activation. Loss of UHRF1 in cancer cells led to significant reduction of tumor masses in a syngeneic lung cancer model. The tumor shrinkage was due to increased infiltration of T cells with the enhanced ability to proliferate and produce IFN-gamma. Mechanistically, lung cancer cells deficient of UHRF1 had elevated expression of endogenous retrovirus genes due to DNA demethylation, which associated with enhanced type I interferon responses. Type I interferons are potent antitumor effector molecules by promoting immune cell infiltration into the tumor microenvironment, yet they can also dampen T cell activation by upregulating PD-L1 expression in tumor cells. Surprisingly, UHRF1 deficient cancer cells failed to upregulate PD-L1 even in the presence of excessive type I interferons, indicative of additional regulatory pathways for PD-L1. We further found that UHRF1 directly interacted with PD-L1 promoter regions and that UHRF1 promoted PD-L1 transcription. Therefore, the inhibition of UHRF1 augmented type I interferon responses while diminished PD-L1 expression, which synergistically contributed to the immune activation phenotype. Moreover, in non-small cell lung cancer (NSCLC) patients, UHRF1 expression was positively correlated with PD-L1 expression as well as poor prognosis. Together, our data suggested that UHRF1 represents a novel immuno-oncology target for NSCLC.

Citation Format: Handan Xiang, Junying Chen, Xiao-Fan Wang. UHRF1 deficiency exerts anti-tumor immune responses in non-small cell lung 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 3059. doi:10.1158/1538-7445.AM2017-3059
Abstract 3070: Lenalidomide and SMAC mimetic LCL161 show combination activity in cells harboring loss of chromosome 5q
Authors: Hafeez Nafeeza , Gaither Alex , Porter Dale
Published: July 2017

LCL161 is a small molecule SMAC mimetic that binds to cIAP1, cIAP2, and XIAP and sensitizes cancer cells to TNF-alpha as well as other inducers of cell death. An siRNA screen was performed in the SK-OV-3 cancer cell line to identify genes that when knocked down prevent LCL161-mediated cell death. As expected, siRNAs targeting RIPK1 rescued cells from LCL161-induced death. Unexpectedly, siRNAs targeting GPR68 also rescued cells from LCL161-induced death. Since knockdown of GPR68 has been reported to rescue myelodysplastic syndrome (MDS)-derived cells harboring loss of chromosome 5q from lenalidomide treatment, we tested for combination activity with lenalidomide plus LCL161 in 7-day in vitro proliferation assays. Interestingly, combination activity was observed in the SET-2 cell line harboring cytogenetic features of MDS including loss of chromosome 5q. These data suggest that, in combination, LCL161 may improve responses to lenalidomide in subsets of MPN and MDS patients and potentially additional contexts where lenalidomide is active.

Citation Format: Nafeeza Hafeez, Alex Gaither, Dale Porter. Lenalidomide and SMAC mimetic LCL161 show combination activity in cells harboring loss of chromosome 5q [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 3070. doi:10.1158/1538-7445.AM2017-3070
Abstract 3116: Dysregulated cholesterol synthesis is a therapeutic vulnerability in glioblastoma
Authors: Kambach Diane M. , Halim Alan S. , Cauer A. Gesine , Sun Qian , Tristan Carlos A. , Celiku Orieta , Kesarwala Aparna H. , Shankavaram Uma , Batchelor Eric , Stommel Jayne M.
Published: July 2017

A hallmark of cellular transformation is the evasion of contact-dependent inhibition of growth. To find new therapeutic targets for glioblastoma, we looked for pathways that are inhibited by high cell density in normal astrocytes but have lost this regulation in glioma cells. Here we report that at high cell density, normal astrocytes turn off cholesterol synthesis but glioma cells keep this pathway on. Correspondingly, cholesterol pathway upregulation is associated with poor prognosis in glioblastoma patients. Densely-plated glioma cells increase oxygen consumption to synthesize cholesterol, resulting in a decrease in reactive oxygen species, TCA cycle intermediates, and ATP, but without a compensating increase in aerobic glycolysis. This constitutive cholesterol synthesis is controlled by the cell cycle, as it can be turned off by cyclin dependent kinase inhibitors and it correlates with disabled cell cycle control though loss of p53 and RB. Finally, glioma cells, but not astrocytes, are sensitive to cholesterol synthesis inhibition downstream of the mevalonate pathway, suggesting that specifically targeting cholesterol synthesis might be an effective treatment for glioblastoma.

Citation Format: Diane M. Kambach, Alan S. Halim, A. Gesine Cauer, Qian Sun, Carlos A. Tristan, Orieta Celiku, Aparna H. Kesarwala, Uma Shankavaram, Eric Batchelor, Jayne M. Stommel. Dysregulated cholesterol synthesis is a therapeutic vulnerability in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3116. doi:10.1158/1538-7445.AM2017-3116
Abstract 3124: Expression and therapeutic implications of cyclin dependent kinase 4 (CDK4) in osteosarcoma
Authors: Zhou Yubing , Hornicke Francis , Duan Zhenfeng
Published: July 2017

Overexpression and/or hyperactivation of cyclin-dependent kinase 4 (CDK4) has been found in many types of human cancers, and a CDK4 specific inhibitor, palbociclib, has been recently approved by the FDA for the treatment of breast cancer. However, the expression and the therapeutic potential of CDK4 in osteosarcoma remain unclear. In the present study, CDK4 was found to be highly expressed in human osteosarcoma tissues and cell lines as compared with normal human osteoblasts. Elevated CDK4 expression correlated with metastasis potential and poor prognosis in osteosarcoma patients as determined by immunohistochemical analysis in a human osteosarcoma tissue microarray (TMA). CDK4 inhibition by either palbociclib or specific small interference RNA (siRNA) exhibited dose-dependent inhibition of osteosarcoma cell proliferation and growth, accompanied by suppression of the CDK4/6-cyclinD-Rb signaling pathway. Flow cytometry analysis showed that CDK4 knockdown arrested osteosarcoma cells in the G1 phase of the cell cycle and induced cell apoptosis. Furthermore, inhibition of CDK4 significantly decreased osteosarcoma cell migration in vitro determined by the wound healing assay. These data highlight that CDK4 may be a potential promising therapeutic target in the treatment of human osteosarcoma.

Citation Format: Yubing Zhou, Francis Hornicke, Zhenfeng Duan. Expression and therapeutic implications of cyclin dependent kinase 4 (CDK4) in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3124. doi:10.1158/1538-7445.AM2017-3124
Abstract 3133: NF-κB-dependent inflammatory responses offset sorafenib cytotoxicity in hepatocellular carcinoma via TIFA
Authors: Wei Tong-You Wade , Wu Pei-Yu , Wu Ting-Jung , Tsai Ming-Daw
Published: July 2017

Sorafenib is the promising first-line drug to treat advanced hepatocellular carcinoma (HCC), with the acquired resistance within 6 months in most treated patients. The epithelial-mesenchymal transition (EMT)-driven cancer metastasis contributes to sorafenib resistance via multiple signaling pathways, but the underlying molecular mechanism remains elusive. Recent studies showed that inflammatory cytokines TNF-α functionally initiates EMT through NF-κB mediated Snail activation in HCC, and that TRAF-interacting protein TIFA sustains the positive feedback loop between TNF-α and NF-κB that enhances chemoresistance in AML. Here we investigated the functional link between TIFA-regulated NF-κB inflammatory signaling and sorafenib resistance as a potential therapeutic target in the treatment of HCC. We showed that severe hepatitis induced in HCC mice promoted intrahepatic metastasis concurrently with NF-κB-driven EMT via Snail, and that HCC patients with hepatitis displayed poorer responses to sorafenib and unfavorable clinical outcome. In support, inflammatory TNF-α stimulation promoted levels of TIFA, NF-κB signaling factors, and EMT axis independent of sorafenib treatment, while silencing of TIFA or RelA perturbed sorafenib-dependent cytokine secretion in HCC cells. In addition, silencing of TIFA or RelA suppressed pro-survival factors Bcl-2 and Bcl-XL, and promoted pro-apoptotic factor BAX in response to sorafenib treatment in vitro. Consequently, HCC cells regained sorafenib chemosensitivity upon silencing of TIFA or RelA through promoted apoptosis. To further explore the therapeutic relevance, shRNAs were delivered using the liver-tropic adeno-associated virus serotype 8 in mice with orthotopic HCC xenografts, and results showed that targeting TIFA or RelA specifically enhanced sorafenib chemotoxicity resulting in more prominent tumor regression. Our results collectively showed that TIFA and NF-κB support inflammatory responses to offset sorafenib cytotoxicity, and that their targeting are therapeutically effective concomitant with sorafenib treatment in HCC.

Citation Format: Tong-You Wade Wei, Pei-Yu Wu, Ting-Jung Wu, Ming-Daw Tsai. NF-κB-dependent inflammatory responses offset sorafenib cytotoxicity in hepatocellular carcinoma via TIFA [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 3133. doi:10.1158/1538-7445.AM2017-3133
Abstract 3157: Osimertinib (AZD9291) is effective against NSCLC cells harboring EGFR exon 20 insertion mutations
Authors: Lee Yusoo , Kim Tae Min , Kim Dong-Wan , Ahn Yong-Oon , Kim Soyeon , Keam Bhumsuk , Kim Miso , Heo Dae Seog
Published: July 2017

Background: Although EGFR exon 20 insertion mutation is the third most common among EGFR-mutant NSCLC, this mutant clones are resistant to the first-generation EGFR TKIs. In addition, the efficacy of the second or third-generation EGFR TKIs is unknown in NSCLC with EGFR exon 20 insertion mutation.

Methods: Various EGFR exon 20 insertion mutations were introduced into Ba/F3 cells as follows: A763_Y764insFQEA in C-helix; and V769_D770insASV, D770_N771insNPG, D770_N771insSVD, P772_H773insPR, H773_V774insNPH, H773_V774insH, and H773_V774insAH in loop following C-helix. The patient-derived SNU-3173 cell line was developed in a patient with EGFRH773_V774insAH mutation. Ba/F3 cells transfected with EGFR exon 20 insertion mutations and SNU-3173 were exposed to the EGFR TKIs (the 1st-generation gefitinib and erlotinib; the 2nd-generation dacomitinib and afatinib; and the 3rd-generation rociletinib, olmutinib, nazartinib, and osimertinib) and cell viability assays were performed. In addition, immunoblotting was performed to evaluate the downstream signals of EGFR pathway.

Results: Cell viability assay showed that EGFR exon 20 insertion mutants are resistant to the 1st-generation EGFR TKIs, except for EGFRA763_Y764insFQEA-mutant Ba/F3 cells. IC50 values of the 2nd-generation EGFR TKIs are five to hundred-folds lower than the 1st-generation EGFR TKIs. The 2nd-generation EGFR TKIs were effective against EGFR exon 20 inserted Ba/F3 cells and down-regulated EGFR downstream signals. The 3rd-generation EGFR mutation-selective inhibitors showed a moderate efficacy except osimertinib that was effective and down-regulated EGFR downstream signals. In addition, SNU-3173 cells were moderately toxic to afatinib (IC50 46.2±5.2nM) as well as osimertinib (IC50 403.2±3.0nM).

IC50 values of EGFR exon 20 insertion mutation Ba/F3 cellsIC50 (nM)FQEAASVSVDNPGPRHAHNPHGefitinib32.7525.7752.71597.01363.02581.02259.01701.0Erlotinib22.3486.9338.3765.7798.21523.01438.0562.1Dacomitinib0.00243.55.715.0205.0608.4556.312.3Afatinib0.0082.344.911.494.7297.7204.425.4Rociletinib219.7367.191.5131.21093.93710.05085.4498.7Olmutinib76.082.195.664.5332.6952.41406.6337.1Osimertinib3.68.18.9128.2247.2128.2880.338.2Nazartinib28.322.336.657.6747.8872.11606.8118.0

Conclusions: Overall, osimertinib showed effectiveness against NSCLC cells with EGFR exon 20 insertion mutations. Our results warrant a clinical trial of osimertinib in NSCLC patients with EGFR exon 20 insertion mutations.

Citation Format: Yusoo Lee, Tae Min Kim, Dong-Wan Kim, Yong-Oon Ahn, Soyeon Kim, Bhumsuk Keam, Miso Kim, Dae Seog Heo. Osimertinib (AZD9291) is effective against NSCLC cells harboring EGFR exon 20 insertion mutations [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 3157. doi:10.1158/1538-7445.AM2017-3157
Abstract 3162: HRAS G12V predicts for innate resistance to PI3Kα inhibition in head and neck squamous cell cancer
Authors: Ruicci Kara M. , Sun Ren , Black Morgan , Pinto Nicole , Yoo John , Fung Kevin , Macneil Danielle , Aillies Lauire , Mymryk Joseph S. , Boutros Paul C. , Barrett John W. , Nichols Anthony C.
Published: July 2017

Introduction: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with an incidence of ~600 000 new cases per year and a 50% mortality rate for advanced disease. The mutational landscape of HNSCC has been recently elucidated, introducing the possibility for targeted therapeutic approaches. PIK3CA—which encodes the α-catalytic subunit of PI3K (PI3Kα)—is the most frequently altered actionable target in HNSCC, however it is not presently clear which patients benefit most from PI3Kα-inhibition. BYL719 is a leading PI3Kα inhibitor in clinical development for HNSCC. We previously examined the responses of a large panel of genetically-characterized HNSCC cell lines to BYL719 and identified activating HRAS G12V mutations as strong predictors for BYL719 resistance. Here we examine if this mutation is able to individually modulate BYL719 response through overexpression and knockdown studies.

Methods: To determine if HRAS G12V was able to modulate BYL719 sensitivity, we knocked down HRAS in HRAS G12V and HRAS wild-type (WT) cell lines using RNA interference, and then treated cells over 10-point dose ranges with BYL719. Sensitivity was determined by calculating IC50 (half-maximal inhibitory concentration) values at 72 hours using PrestoBlue®. Constructs expressing wildtype (WT) HRAS or HRAS G12V were transfected into BYL719-sensitive cells for overexpression studies and cells were selected using G418 Sulfate for 1 month. Proliferation and BYL719 sensitivity (IC50 values) were measured as described. Immunoblotting was used to examine activity of the PI3K-AKT axis with and without BYL719 (1μM) for 24 hours.

Results & Conclusions: HRAS knockdown sensitized HRAS G12V lines to BYL719 (lower IC50), but did not affect the response of WT HRAS cells. WT HRAS and HRAS G12V overexpression significantly increased cellular proliferation and promoted BYL719 resistance. In HRAS G12V cell lines, we observed high levels of active phosphorylated AKT (S473/T308), even in the presence of BYL719. Collectively these findings highlight the predictive role of HRAS G12V for innate BYL719 resistance and contribute to our understanding of which patients may respond best on BYL719 therapy.

Citation Format: Kara M. Ruicci, Ren Sun, Morgan Black, Nicole Pinto, John Yoo, Kevin Fung, Danielle Macneil, Lauire Aillies, Joseph S. Mymryk, Paul C. Boutros, John W. Barrett, Anthony C. Nichols. HRAS G12V predicts for innate resistance to PI3Kα inhibition in head and neck squamous cell 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 3162. doi:10.1158/1538-7445.AM2017-3162
Abstract 3167: MIF-induced stat3 activation promotes resistance to MEK blockade in KRAS mutant colorectal cancer cells
Authors: Cheon Seul-Ki , Kim Hwang-Phill , Park Ye-Lim , Lee Si Hyun , Kang Jun-Kyu , Lim Yoojoo , Song Sang-Hyun , Han Sae-Won , Kim Tae-You
Published: July 2017

Although MEK blockade has been highlighted as a promising anti-tumor drug, it has poor clinical efficacy in KRAS mutant colorectal cancer. Several feedback systems have been described in which inhibition of one intracellular pathway leads to activation of a parallel signaling pathway, thereby decreasing the effectiveness of single-MEK targeted therapies. In this study, we describe a feedback mechanism in which MEK inhibition leads to activation of macrophage migration inhibitory factor (MIF)-induced stat3 signaling pathway in KRAS mutant colorectal cancer (CRC) cells. We found that KRAS mutant CRC cells with refametinib, MEK inhibitor, induced MIF secretion and resulted in activation of Stat3. MIF knockdown by siRNA partially restored sensitivity to refametinib in KRAS mutant cells. In addition, combination with refametinib and 4IPP, a MIF inhibitor, effectively reduced the activity of stat3 and MAPK, more than single agent treatment. As a result, combined therapy was found to exhibit a synergistic growth inhibitory effect against refametinib-resistant cells by downregulating MIF expression. These results reveal that MIF-induced stat3 activation evoked an intrinsic resistance to refametinib. Our results provide the basis for a rational combination strategy against KRAS mutant colorectal cancers, predicated on the understanding of cross-talk between the MEK and MIF pathways.

Citation Format: Seul-Ki Cheon, Hwang-Phill Kim, Ye-Lim Park, Si Hyun Lee, Jun-Kyu Kang, Yoojoo Lim, Sang-Hyun Song, Sae-Won Han, Tae-You Kim. MIF-induced stat3 activation promotes resistance to MEK blockade in KRAS mutant colorectal cancer cells [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 3167. doi:10.1158/1538-7445.AM2017-3167
Abstract 3171: Analysis of "drug addiction" mechanisms in the drug-resistant ROS1-rearranged cancer cells
Authors: Ogura Hayato , Adachi Jun , Tomonaga Takeshi , Fujita Naoya , Katayama Ryohei
Published: July 2017

ROS1-rearranged non-small-cell lung cancer (NSCLC) is observed in approximately 1% of lung cancer patients. Crizotinib, ALK/ROS1/cMET tyrosine kinase inhibitor (TKI), showed high effect against ROS1-rearranged NSCLC in the clinical trials and is approved in US and EU. However, the cancers inevitably relapse due to the acquired resistance such as ROS1-G2032R mutation. We previously reported that cabozantinib could overcome those secondary mutations mediated crizotinib-resistance. Based on this finding, we tried to establish cabozantinib-resistant ROS1 mutant cells by ENU mutagenesis screening. Surprisingly, we found out a few CD74-ROS1 mutant clones that only grew in the presence of low-dose cabozantinib. Namely, these mutant clones were “addicted” to cabozantinib. When the fluorescent labeled cabozantinib addicted ROS1 mutant cells were co-cultured with non-addicted CD74-ROS1-WT-BaF3 cell in the presence or absence of low-dose cabozantinib, the drug-addicted cells became dominant in the low-dose cabozantinib, and CD74-ROS1-WT cells became dominant in the absence of it. To analyze the molecular mechanism of “drug-addiction” in the addicted CD74-ROS1 mutant cells, we performed comprehensive analysis such as inhibitor screening, cDNA microarray, and phosphoproteome analysis. As the result, we observed that the expression and phosphorylation of CD74-ROS1 in the addicted cells were excessively upregulated within 24 hours after removal of cabozantinib or other ROS1 inhibitors, such as crizotinib or lorlatinib. We also observed that the apoptosis was induced in these cells upon ROS1 inhibitor removal, whereas ROS1 mediated growth signaling were simultaneously activated. Consistent with this, cDNA microarray analysis revealed that the expression of both cell survival and cell death related genes were changed by ROS1 inhibitor removal. Furthermore, we identified that several proteins were highly phosphorylated by excessive ROS1 activation originated from ROS1 inhibitor removal. These observations suggest that these drug-addicted cells were died by excessive oncogenic signaling, and the appropriate oncogene signaling by low-dose TKI made them survive and grow without inducing apoptosis. Thus, the excessive oncogene signaling has “double-edged sword” characteristics for cancer cell viability and those characteristics could be a new therapeutic target.

Citation Format: Hayato Ogura, Jun Adachi, Takeshi Tomonaga, Naoya Fujita, Ryohei Katayama. Analysis of "drug addiction" mechanisms in the drug-resistant ROS1-rearranged cancer cells [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 3171. doi:10.1158/1538-7445.AM2017-3171
Abstract 3175: Context-dependent role of ARF for response to chemotherapy in muscle invasive bladder cancer
Authors: Rong Lijie , Owczarek Tomasz , Kobayashi Takashi , Abate-Shen Cory
Published: July 2017

Muscle invasive bladder cancer (MIBC) is a significant cause of cancer death, in part because there are relatively few treatments and these have limited efficacy. Our studies show that expression of 14ARF (aka ARF), one of the major transcripts encoded by the CDKN2A tumor suppressor locus, is frequently up-regulated in MIBC, particularly in more aggressive disease subtypes. Our results further show that accumulation of ARF protein in the nucleolus is associated with poor outcome and reduced response to chemotherapy. Using both genetically-engineered mouse (GEM) models and human xenograft models of MIBC, we demonstrate that tumors expressing ARF display poor response to treatment with the platinum-based chemotherapy agent cisplatin. This is mediated in part by expression of the integrin-binding protein ITGB3BP (CENPR) and reflects ARF-dependent inhibition of protein translation, which is attenuated by drug treatment. Our findings reveal an unexpected context-dependent role for ARF in modulating drug response in bladder cancer through its ability to regulate protein translation.

Citation Format: Lijie Rong, Tomasz Owczarek, Takashi Kobayashi, Cory Abate-Shen. Context-dependent role of ARF for response to chemotherapy in muscle invasive bladder 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 3175. doi:10.1158/1538-7445.AM2017-3175
Abstract 3190: Using a genome-wide shRNA screen to investigate the mechanism of stroma-conferred gemcitabine resistance in pancreatic ductal adenocarcinoma
Authors: Mills Graham D. , Erard Nicolas , Richards Frances M. , Hannon Gregory J. , Jodrell Duncan I.
Published: July 2017

The purpose of this study is to identify mechanisms of resistance to gemcitabine in pancreatic ductal adenocarcinoma (PDAC) using an in vitro coculture system. The inherent resistance of PDAC to first line treatments such as gemcitabine has been attributed to the dense fibrotic stroma. Targeting stromal components, in particular cancer-associated fibroblasts (CAFs), the activated form of pancreatic stellate cells (PSCs), is a promising avenue for therapeutic intervention. We have demonstrated previously the induction of gemcitabine resistance when co-culturing KRAS G12D; p53 R172H; Pdx-cre (KPC) mouse PDAC-derived tumour cells with αSMA+ fibroblast-like cells from PDAC tumour (FLCs), in comparison to PDAC cells co-cultured with PSCs or in monoculture. This resistance-inducing effect is specific to cells of mesenchymal phenotype, is transient in nature and a product of cell-cell contact. To investigate the exact mechanism by which PDAC cells become resistant to gemcitabine in coculture, we undertook a genome-wide shRNA depletion viability screen, infecting KPC mouse PDAC cells with a pooled shERWOOD UltramiR shRNA library both in mono- and co-culture with FLCs, in the presence and absence of gemcitabine. Next-generation sequencing and differential expression analysis, using DESeq2, identified shRNAs depleted through 6 cycles of gemcitabine treatment in coculture, indicating they target genes driving the resistance effect. Pathway analysis of differential expression using MetaCore implicated DNA damage repair and protein modification pathways in the gemcitabine resistance effect. Depletion of p97, ATR, or Chk1 all sensitised PDAC cells to gemcitabine in coculture, corroborating findings with translational value in previous studies. Further evaluation is ongoing using validation shRNA dropout assays in combination with targeted literature reviews to collate a candidate list of targetable genes with translational value in preventing and combating chemotherapy resistance in patients with PDAC.

Citation Format: Graham D. Mills, Nicolas Erard, Frances M. Richards, Gregory J. Hannon, Duncan I. Jodrell. Using a genome-wide shRNA screen to investigate the mechanism of stroma-conferred gemcitabine resistance in pancreatic ductal adenocarcinoma [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 3190. doi:10.1158/1538-7445.AM2017-3190