bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2347: High-throughput scRNA-seq analysis using Celsee Nano-prep 250K sequencing chip on Celsee C-PREP GENESIS
Authors: Sharma Vishal Premdev , Chow William Chiro , Boniface Brian , Gleason Kyle , Payne Austin , Gogoi Priya , Handique Kalyan
Published: August 2018

For effective targeted therapeutics, it is critical to identify cancer mutations in cells that are often regional and heterogenic. Recent advances in single-cell mRNA sequencing (scRNA-seq) have made it possible to study intratumor heterogeneity and its role in cancer progression, metastasis, and treatment resistance. Next-generation sequencing (NGS)-based studies have identified critical genetic alterations in a variety of malignancies. However, the current workflow of existing technologies leads to significant loss of cells and thus loss of critical information. In this study we present a technology that decreases cell loss and increases efficiency of scRNA-seq. C-PREP GENESIS is a fully automated, random-access, multiple-sample processor for single-cell sequencing preparation. C-PREP GENESIS allows automated fluid dispensing and controlled pumping through Nanochips. The instrument utilizes unitized reagent cartridge for dispensing reagents for wash, lysis, cDNA synthesis and oil (for partitioning). With its automated thermocycling (4oC- 100oC), it can achieve the single cell to cDNA synthesis on beads with unique cell barcode and UMI tags in under 2 hours with an efficiency of ~80% of the 50,000 single cells captured on the nano-PREP 250K sequencing chip. The cDNA can then be retrieved from the chip for downstream library preparation. Alternatively, users can perform a targeted library prep PCR on the same instrument. Here we present scRNA-seq analysis of single cells obtained from patient tumor biopsy samples and cancer tissue culture single cells to discuss the critical mutations observed in single cells of the heterogenic tumor populations.

Citation Format: Vishal Premdev Sharma, William Chiro Chow, Brian Boniface, Kyle Gleason, Austin Payne, Priya Gogoi, Kalyan Handique. High-throughput scRNA-seq analysis using Celsee Nano-prep 250K sequencing chip on Celsee C-PREP GENESIS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2347.
Abstract 2356: Prioritizing cancer gene drivers defined in large-scale cancer genome analyses using the SBCDDB
Authors: Newberg Justin Y. , Mann Michael B. , Mann Karen M.
Published: August 2018

Next-generation sequencing approaches to identify molecular signatures of cancer have uncovered few frequently altered genes, many infrequently altered genes, and various transcriptional changes associated with cancer initiation/progression. Prioritization of genetic and epigentic alterations in various contexts is therefore important for biological discovery and therapy development. Comparative oncogenomics is a powerful approach to identify drivers that impact cancer development. Importantly, data integration across different experimental systems and data types is imperative to the success of these oncogenomics approaches. Sleeping Beauty (SB) insertional mutagenesis coupled with next generation sequencing forms a powerful cancer gene discovery framework widely used to identify and prioritize candidate drivers in mouse models of human cancers. SB has been used to model over 20 different human cancers and identify cooperating drivers underlying tumor development. Integration of SB data with human cancer genomic and transcriptomic data from The Cancer Genome Atlas (TCGA) / Genomic Data Commons (GDC) is one way to identify and prioritize genes for biological validation and targeted therapies. Our group has built a resource, the Sleeping Beauty Cancer Driver Database (SBCDDB), to identify sets of cancer driver genes associated with specific tumor types or genetic aberrations underling cancer initiation. Using the SBCDDB, in combination with human mutation, copy-number alteration, and mRNA-seq datasets from the Broad Firehose, we have prioritized infrequently mutated driver genes in human tumors that are recurrently altered in SB tumors. Cancer drivers defined in both human tumors and in related mouse models of cancer, using distinct but complimentary methods to discover and catalog mutations, provide strong evidence for a causal role of detectable alteration in tumor initiation and/or progression. This work is an important step in a larger, comparative pan-cancer approach to better define molecular signatures that make up the tumor landscape.

Citation Format: Justin Y. Newberg, Michael B. Mann, Karen M. Mann. Prioritizing cancer gene drivers defined in large-scale cancer genome analyses using the SBCDDB [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2356.
Abstract 2359: Adults with heterozygote carrier of immune deficiency variants have increased risk of cancer
Authors: Kim Daeyoon , Choi Murim , Seong Moon-woo , Yoon Sung-Soo , Koh Youngil , Yun Hongseok
Published: August 2018

Introduction: Relationship between several mendelian disorders and cancer has been reported. Although correlation between immune-deficiency disorder and cancer development is well known, most studies have been conducted on patients with established immunodeficient diseases. Rate of tumor development in recessive heterozygous carriers of immunodeficiency genes is not clear yet.

Methods: Using whole-genome sequence data from International Cancer Genome Consortium (ICGC) Pan-Cancer Analysis of Working Group (PCAWG) and 1000 Genome projects, we analyzed 110 genes which are associated with immunodeficiency disorder and cancer risk. The 110 genes are reported previously as pathogen of immunodeficiency disorder and present in OMIM database. We selected a pathogenic variant of the 110 genes based on the clinical database such as HGMD and Clivar.

Results: Total of 461 pathogenic variants were detected in both cohorts. The prevalence of pathogenic variants of Immunodeficiency disorder was 4.7% in the ICGC-PCAWG cohort and 3.3% in 1000 Genome cohort. Pathogenic variants were observed more frequently in ICGC-PCAWG than in 1000 Genome cohort (p=0.00267) (odds ratio 1.55, 95% confidence interval 1.16-2.06), implying that immune deficiency disorder increases cancer risk. Gallbladder cancer patients most frequently had pathogenic variants (25%, 3/12) followed by ovarian cancer patients (8%, 9/110). The gene with the strongest cancer-evoking potential was TYK2 (p= 4.978 × 10-7). In addition, seven genes were uncovered to be significantly associated with cancer development (RORC, ATM, RTEL1, CTSC, RAG1, PMS2 and IRAK4).

Conclusion: In conclusion, using large whole-genome sequencing global projects, we statistically prove the previous notion that immunodeficiency provokes cancer development. We identified specific germline variants associated with cancer, which would be considered as cancer-predisposing genes.

Citation Format: Daeyoon Kim, Murim Choi, Moon-woo Seong, Sung-Soo Yoon, Youngil Koh, Hongseok Yun. Adults with heterozygote carrier of immune deficiency variants have increased risk of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2359.
Abstract 2362: Comprehensive portrait of canonical and non-canonical splicing in cancer
Authors: Jayasinghe Reyka G. , Cao Song , Gao Qingsong , Wyczalkowski Matthew A. , Sengupta Sohini , Walter Matthew J. , Maher Christopher , Wendl Michael C. , Chen Feng , Eyras Eduardo , Lazar Alexander J. , Chen Ken , Shmulevich Ilya , Ding Li , Group The Splicing Analysis Working
Published: August 2018

Current annotation methods typically classify mutations as disruptors of splicing if they fall on either the consensus intronic dinucleotide splice donor, GT, or the splice acceptor, AG. As a group, splice site mutations have been presumed to be invariably deleterious because of their disruption of the conserved sequences that are used to identify exon-intron boundaries. While this classification method has been useful, increasing evidence suggests that mutations outside of the canonical splice site can lead to transcriptional changes beyond disruption of the nearest junction. In this study, we have developed the MiSplice pipeline to determine the local and global effects of genomic mutations on splicing across 33 cancer types. To evaluate the local effects of mutations on splicing, we applied MiSplice to identify splice-disrupting mutations (SDMs) and splice-creating mutations (SCMs), genome-wide. We identified 1,964 novel SCMs, of which 26% and 11% were mis-annotated as missense and silent mutations and validated 10 of 11 genes in a mini-gene splicing assay. SDM identification predicted complex splicing patterns associated with canonical splice site mutations and identified a handful of mutations in proximity to the canonical junction that disrupt splicing factor binding sites. Interestingly, further investigation of the novel neoantigens produced by SCMs and SDMs are likely several folds more immunogenic than missense mutations. To explore the global impact of mutations on splicing we focused on recurrent and adjacent mutations disrupting the spliceosomal complex and related splicing factor genes from over 400 curated splice related genes. In addition, we applied HotSpot3D to identify splice-factor mutations (SFMs) that are significantly proximal to one another. Our analysis has identified novel SFMs that disrupt the spliceosomal complex and globally impact downstream splicing targets creating novel peptide sequences and alter key cancer genes. The current study has greatly extended the insight into the transcriptional ramifications of genomic alterations by integrating DNA and RNA sequencing data and painting the portrait of alternative splicing across cancer genomes.

Citation Format: Reyka G. Jayasinghe, Song Cao, Qingsong Gao, Matthew A. Wyczalkowski, Sohini Sengupta, Matthew J. Walter, Christopher Maher, Michael C. Wendl, Feng Chen, Eduardo Eyras, Alexander J. Lazar, Ken Chen, Ilya Shmulevich, Li Ding, The Splicing Analysis Working Group. Comprehensive portrait of canonical and non-canonical splicing in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2362.
Abstract 2370: miR-19b enhances proliferation and apoptosis resistance via the epidermal growth factor receptor signalling pathway by targeting PP2A and BIM in non-small cell lung cancer
Authors: Vassella Erik
Published: August 2018

Background: Epidermal growth factor receptor (EGFR) mutations enable constitutive active downstream signaling of PI3K/AKT, KRAS/ERK and JAK/STAT pathways, and promote tumor progression by inducing uncontrolled proliferation, evasion of apoptosis and migration of non-small cell lung cancer (NSCLC). In addition, EGFR mutations increase the susceptibility of patients with NSCLC to tyrosine kinase inhibitor (TKI) therapy, but treated patients will invariably relapse with resistant disease. A global understanding of underlying molecular mechanisms of EGFR signaling may improve the management of NSCLC patients. Results: Here, we defined 17 miRNAs that are regulated by the PI3K/AKT branch of the EGFR signaling pathway. Bioinformatics analysis revealed that miRNAs that are dysregulated following PI3K/AKT inhibition act in a concerted manner to enhance the activity of the EGFR signaling pathway. These findings were closely mirrored by attenuation of miR-19b expression in NSCLC cell lines, where cell cycle progression, clonogenic growth and migration were reduced and apoptosis was enhanced. Conversely, miR-19b overexpression resulted in enhanced phosphorylation of ERK, AKT and STAT in EGFR mutant NSCLC cells. Co-treatment of NSCLC cells with the tyrosine kinase inhibitor (TKI) gefitinib and anti-miR-19b construct reduced migration and clonogenic growth in a synergistic manner suggesting that EGFR and miR-19b act together to control oncogenic processes. Serine/threonine phosphatase PP2A subunit PPP2R5E and BCL2L11 encoding BIM were identified as major targets of miR-19b. Consistent with this finding, PP2A activity was strongly enhanced in NSCLC transduced with anti-miR-19b, but not in cells co-transduced with anti-miR-19b and shPPP2R5E, suggesting that PPP2R5E is a major constituent of the PP2A complex. Accordingly, proliferation enhancement by miR-19b was due to targeting PPP2R5E. In contrast, apoptosis resistance was mainly due to targeting BCL2L11. Conclusion: Our results provide insight into the importance of targeting PPP2R5E and BCL2L11 by miR-19b in oncogenic processes of NSCLC. Attenuation of miR-19b expression could potentially be exploited in adjuvant therapy of EGFR mutant NSCLC.

Citation Format: Erik Vassella. miR-19b enhances proliferation and apoptosis resistance via the epidermal growth factor receptor signalling pathway by targeting PP2A and BIM in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2370.
Abstract 2373: ADA-07 suppresses solar ultraviolet-induced skin carcinogenesis by directly inhibiting TOPK
Authors: Zhang Tianshun , Wang Qiushi , Gao Ge , Ann Bode , Dong Zigang
Published: August 2018

Cumulative exposure to solar ultraviolet (SUV) irradiation is regarded as the major etiologic factor in the development of skin cancer. The activation of the mitogen-activated protein kinase (MAPK) cascades occurs rapidly and is vital in the regulation of SUV-induced cellular responses. The T-LAK cell-originated protein kinase (TOPK), an upstream activator of MAPKs, is heavily involved in inflammation, DNA damage, and tumor development. However, the chemopreventive and therapeutic effects of specific TOPK inhibitors in SUV-induced skin cancer have not yet been elucidated. In the current study, ADA-07, a novel TOPK inhibitor, was synthesized and characterized. Pull-down assay results, ATP competition and in vitro kinase assay data revealed that ADA-07 interacted with TOPK at the ATP-binding pocket and inhibited its kinase activity. Western blot analysis showed that ADA-07 suppressed SUV-induced phosphorylation of ERK1/2, p38, and JNKs, and subsequently inhibited AP-1 activity. Importantly, topical treatment with ADA-07 dramatically attenuated tumor incidence, multiplicity, and volume in SKH-1 hairless mice exposed to chronic SUV. Our findings suggest that ADA-07 is a promising chemopreventive or potential therapeutic agent against SUV-induced skin carcinogenesis that acts by specifically targeting TOPK.

Citation Format: Tianshun Zhang, Qiushi Wang, Ge Gao, Bode Ann, Zigang Dong. ADA-07 suppresses solar ultraviolet-induced skin carcinogenesis by directly inhibiting TOPK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2373.
Abstract 2375: Concurrent HER or PI3K inhibition potentiates the anti-tumor effect of ERK inhibitor BVD-523 (ulixertinib) in preclinical pancreatic cancer models
Authors: Jiang Hongmei , Xu Mai , Li Lin , Highkin Maureen , Zhang Daoxiang , Li Qiong , Wang-Gillam Andrea , Lim Kian-Huat
Published: August 2018

To date, effective treatments for inoperable pancreatic ductal adenocarcinoma (PDAC) remain elusive. Targeting KRAS, the gene that is mutated in >95% of PDAC, is a heavily pursued strategy, but unsuccessful in the clinic. Therefore, targeting key effector cascades of KRAS oncoprotein, particularly the mitogenic RAF-MEK-ERK represents the next best strategy. However, RAF or MEK inhibitors have not shown promising clinical efficacy in PDAC. Several studies have shown that cancer cells treated with RAF or MEK inhibitors adopt multiple mechanisms to re-activate ERK signaling. Therefore, development of ERK-specific inhibitors carries the promise to effectively abrogate this pathway. BVD-523 (ulixertinib) is a first-in-class ERK-specific inhibitor that has demonstrated anti-tumor activity in clinical trials. In this study, we showed that BVD-523 effectively inhibits transformed growth of multiple PDAC lines and potentiates the cytotoxic effect of gemcitabine. Moreover, using reverse-phase protein array analysis, we identified potential mechanisms by which PDAC cells may adopt to tolerate ERK inhibition. On this basis, we proposed and tested two rational combinatorial approaches with BVD-523 that showed promising preclinical efficacy in vitro and in mouse xenografts. Overall, we provide the first evidence that PDAC cells may readily adapt to pharmacologic ERK inhibition, and provide combinatorial therapeutic strategies that could benefit patients.

Citation Format: Hongmei Jiang, Mai Xu, Lin Li, Maureen Highkin, Daoxiang Zhang, Qiong Li, Andrea Wang-Gillam, Kian-Huat Lim. Concurrent HER or PI3K inhibition potentiates the anti-tumor effect of ERK inhibitor BVD-523 (ulixertinib) in preclinical pancreatic cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2375.
Abstract 2376: AXL receptor tyrosine kinase stimulates cell softening and cancer progression via Rac signaling pathway
Authors: Suganuma Masami , Iida Keisuke , Yokoyama Shota , Rawangkan Anchalee , Namiki Kozue , Wongsirisin Pattama
Published: August 2018

Cancer cells exhibit different biophysical properties, such as cell stiffness and elasticity, compared with normal cells. It is now well accepted that cell stiffness measured by atomic force microscope (AFM) is a quantitative indicator of cancer progression and metastasis. To study the role of cell softening in malignant progression, Using Transwell assays and AFM, six human non-small cell lung cancer cell lines were classified into two groups: a high motility-low stiffness (HMLS) group and a low motility-high stiffness (LMHS) group. We found a significant difference in expression of the AXL receptor tyrosine kinase, which belongs to the TAM (Tyro3, AXL, Mer) family, in the stimulation of motility and cell softening. HMLS cells expressed higher AXL and phosphorylated AXL levels than LMHS cells. Exogenous AXL gene expression in H1703 LMHS cells exhibited increased motility and decreased stiffness, along with lower levels of actin stress fibre formation. Conversely, the AXL-specific inhibitor R428 and AXL-targeting siRNA reduced motility and increased stiffness in H1299 HMLS cells. Knockdown of AXL gene stimulated both actin stress fibre formation and focal adhesion kinase phosphorylation, which inhibited tumour formation in a mouse xenograft model. Treatment with Ras/Rac inhibitor SCH 51344, which blocks disruption of actin stress fibres, exerted similar effects to AXL inactivation in H1299 and H1703-AXL cells, but not in H1703 cells (no AXL expression). Furthermore, treatment with an ROCK inhibitor, Y27632, did not show any effect in two AXL expressing cells, while it stimulated motility in H1703 cells. Considering all these results, we propose that the Ras/Rac pathway operates downstream of AXL and suppresses the Rho/ROCK pathway. Cell softening induced by AXL activation is a key biophysical property of cancer cells, and AXL is thus a new target for inhibition of malignant progression in non-small cell lung cancer.

Citation Format: Masami Suganuma, Keisuke Iida, Shota Yokoyama, Anchalee Rawangkan, Kozue Namiki, Pattama Wongsirisin. AXL receptor tyrosine kinase stimulates cell softening and cancer progression via Rac signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2376.
Abstract 2379: PTPRT pseudo-phosphatase domain is a denitrase that contributes to its tumor suppressor function
Authors: Zhao Yiqing , Wang Zhenghe
Published: August 2018

Department of Genetics & Genome Sciences and Case Comprehensive Cancer Center, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.

In addition to phosphorylation, tyrosine residues in proteins can have nitration at the 3-carbon position on the phenol ring (Y-NO2). Protein tyrosine nitration occurs under both physiological and pathologic conditions. However, enzymes that add or remove this protein modification remain to be identified. Protein tyrosine phosphatase receptor T (PTPRT) is one of the twelve receptor protein tyrosine phosphatases (RPTPs) that have two catalytic PTP domains in their intracellular parts. While the membrane proximal PTP domains (D1) are protein tyrosine phosphatases, it has been thought that the C-terminal PTP domains (D2) are pseudo-phosphatase that lack enzymatic activity. Here we report that the pseudo-phosphatase domain (D2) of PTPRT is a denitrase that removes nitro-groups from tyrosine residues in paxillin. PTPRT normally functions as a tumor suppressor and is frequently mutated in a variety of human cancers including colorectal cancer. Interestingly, a fifth of tumor-derived mutations of PTPRT are located in the D2 pseudo-PTP domain. We demonstrate that some of the tumor-derived mutations located in the pseudo-phosphatase domain impair the denitrase activity. Moreover, PTPRT mutant mice that inactivate the denitrase activity are susceptible to carcinogen-induced colon tumor formation. Our study uncovers a novel enzyme that functions as a tumor suppressor.

Citation Format: Yiqing Zhao, Zhenghe Wang. PTPRT pseudo-phosphatase domain is a denitrase that contributes to its tumor suppressor function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2379.
Abstract 2385: Evaluation of GCN2 chemical probes: Activity in leukemia
Authors: Lough Lea , Sherman Dan , Ni Eric , Thandapani Palaniraja , Aifantis Ioannis , Cardozo Timothy
Published: August 2018

Cellular stress signals activate adaptive signaling pathways of the mammalian integrated stress response (ISR), which converge at the phosporylation of eIF2α. Drug-like chemical inhibitors targeting major ISR kinases have been identified, with the exception of GCN2. We synthesized and evaluated a series of GCN2 inhibitors based on a triazolo[4,5-d]pyrimidine scaffold. Several compounds potently inhibited GCN2 in vitro in orthogonal assays and displayed good selectivity over the related kinases PERK, HRI, and IRE1. The compounds had low solubility at pH 7.4, but sufficient permeability to inhibit phosporylation of eIF2α in HEK293T cells with an IC50 < 150 nM. In a screen of the NCI-60 cancer cell line panel, the leukemia cells were the only group of cancer cell lines to uniformly exhibit growth inhibition. Given the previously reported genetic association of Gcn2 with resistance to asparaginase, which is a first line clinical leukemia therapy, pharmacologic inhibition of Gcn2 may overcome resistance to asparaginase treatment in leukemia.

Citation Format: Lea Lough, Dan Sherman, Eric Ni, Palaniraja Thandapani, Ioannis Aifantis, Timothy Cardozo. Evaluation of GCN2 chemical probes: Activity in leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2385.
Abstract 2390: When MLK3 meets PAK1: Its implication in breast cancer tumorigenesis
Authors: Das Subhasis , Nair Rakesh Sathish , Mishra Rajakishore , Bhowmik Tanmoy , Rana Basabi , Rana Ajay
Published: August 2018

Mixed Lineage Kinase-3, also known as MAP3K11 is a member of a larger family of MAP3Ks, called MLKs. MLK3 has been reported to play an important role in pro-apoptotic signaling, however its role in tumorigenesis is still not fully elucidated. Previously, we reported that MLK3 is down regulated by estrogen and HER2 amplification and this down regulation provided a survival advantage to ER+ and HER2+ breast cancer cells. To further understand the role of MLK3 signaling in breast cancer pathogenesis, we aimed to identify the upstream kinase(s) that might regulate MLK3 activity and perhaps tumorigenesis.

Based on available information from yeast, where Ste20 members are upstream of Ste11 (i.e. MAP3K), we hypothesized that mammalian homologue of Ste20, PAK1 could be upstream of MLK3. PAK1 kinase has been reported to be a major oncogene in many cancers, including breast cancer. Our results were however, counterintuitive to our hypothesis. We observed that instead, MLK3 was able to associate, and activate PAK1 Kinase activity. These results suggested that perhaps MLK3 is either upstream or parallel to PAK1 in the mammalian signaling network. We also observed that PAK1 was phosphorylated by MLK3 on Ser133 and Ser204 sites. Interestingly, Alanine substitution of S133 site constitutively activated PAK1 kinase activity, whereas Alanine substitution of the S204 site completely blocked MLK3-induced PAK1 activation. Constitutive active MLK3 was not able to phosphorylate and activate double mutant PAK1 S133A, S204A in vitro or in HEK293 cells, clearly indicated that PAK1 S204 site is important for MLK3-induced PAK1 activation. Since PAK1 is an oncogene in breast cancer, we determined the biological effect of S204 phosphorylation on breast cancer progression. Stable breast cancer cell line, MDA-MB-468, expressing PAK1 S204A, inhibited migration, invasion and showed significantly smaller tumors, compared to WT PAK1-expressing cells. Taken together, our results suggest that MLK3-induced modulation of PAK1 activity plays a major role in breast cancer tumorigenesis.

First two authors are equally contributed

Citation Format: Subhasis Das, Rakesh Sathish Nair, Rajakishore Mishra, Tanmoy Bhowmik, Basabi Rana, Ajay Rana. When MLK3 meets PAK1: Its implication in breast cancer tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2390.
Abstract 2392: Evaluation of synergistic PI3K/Akt and MEK5/ERK5 inhibition in triple-negative breast cancer
Authors: Wright Thomas D. , Raybuck Christopher , Gartland Nathan , Wendekier Katy , Monlish Darlene , Chakrabarty Suravi , Flaherty Patrick T. , Burow Matthew E. , Cavanaugh Jane E.
Published: August 2018

Triple negative breast cancers (TNBCs) represent 15-20% of all breast cancers and are often associated with a poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Therefore, there is a need to identify survival pathways that may be targeted in TNBCs. Aberrations in the Phosphoinositide-3-kinase (PI3K) and Mitogen Activated Protein Kinase (MAPK) pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. The crosstalk between PI3K/Akt and MEK1/2/ERK1/2 has been characterized in several systems. However, new evidence suggests that MEK5/ERK5, a member of the MAPK family, is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/Akt and MEK5/ERK5 in three TNBC cell lines: MDA-MB-231, BT549, and MDA-MB-468. We achieved Akt inhibition by using the clinically relevant inhibitors MK-2206 and Ipatasertib. We used a novel compound synthesized in our lab, SC-1-181, and the research tool, XMD8-92, to inhibit MEK5 and ERK5, respectively. Our results indicate that the dual inhibition strategy was more effective than single inhibition due to the loss of crosstalk between the two pathways. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Interestingly, this signaling pattern was observed without disturbing the ERK1/2 pathway. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. Additionally, the dual inhibition strategy was determined to be synergistic in TNBCs and was nontoxic in non-neoplastic cell lines. Lastly, we evaluated the contributions of Bromodomain 4 (BDR4), an off-target effect of XMD8-92, to the synergy mechanism. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.

Citation Format: Thomas D. Wright, Christopher Raybuck, Nathan Gartland, Katy Wendekier, Darlene Monlish, Suravi Chakrabarty, Patrick T. Flaherty, Matthew E. Burow, Jane E. Cavanaugh. Evaluation of synergistic PI3K/Akt and MEK5/ERK5 inhibition in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2392.
Abstract 2401: Effects of carcinoma-associated fibroblasts on cancer metabolism
Authors: Nguyen Tu , Kirsch Jimmy , Nabi Karim , Sazeides Christos , Quinones Addison , Tan Jessica , Antonio Marjorie , Camelo Felipe , Jung Jin , Le Anne
Published: August 2018

Rationale: In previous studies, it has been found that cancer-associated fibroblasts (CAFs) interaction with carcinoma cells and helped cancer cell grow which contributed to cancer progression and aggressiveness. However, how CAFs helped cancer cells grow, specifically what nutrition CAFs provides to tumors has not yet been fully understood.

Objectives: We are studying cancer metabolism to identify pathways to target for cancer therapy. Therefore, in this study, we sought to determine which key metabolites in glucose metabolism, specifically glycolysis and the tricarboxylic acid (TCA) cycle metabolites that CAFs feed cancer cells in order for pancreatic cancer cells (P198), to grow better.

Measurement Methods and Main Results: We first assessed pancreatic cancer cell proliferation (P198 cells) with and without the presence of CAFs through the co-culture technique. We assessed P198 cell number after the co-culture. We found that P198 cells grew better when co-cultured with CAFs as compared to P198 that was grown alone. We next investigated which metabolites produced from the CAFs cells are feeding P198 cells. In order to determine which metabolites from glucose were provided from CAFs to P198 cells, we first grew CAFs in 13C6-labeled glucose, removed the labeling medium, then co-cultured P198 with CAFs. The collected metabolites were assessed using Agilent 6520 Quadrupole-Time-of-Flight (Q-TOF) mass spectrometer with Agilent 1260 HPLC and a 6490 triple-quadrupole (QQQ) mass spectrometer. Metabolites were identified by retention time using in-house compound standard databases. In parallel, metabolites were identified using MS/MS fragmentation data under identical conditions. Metabolic pathways were reconstructed and analyzed in three categories: bioenergetics, biosynthesis and redox homeostasis of glucose metabolism to study these metabolic pathways exchanged between CAF and P198. Metabolite peak intensities were normalized to the protein concentration and cell number. We observed an increase in intensities of metabolites in the TCA cycle and glycolysis in P198 after co-culture with CAFs.

Conclusion: These data supports growth benefits of pancreatic cancer in the presence of CAFs. We also found evidence of the metabolic exchange between CAFs and pancreatic cancer cells, which potentially explains the growth benefit.

Citation Format: Tu Nguyen, Jimmy Kirsch, Karim Nabi, Christos Sazeides, Addison Quinones, Jessica Tan, Marjorie Antonio, Felipe Camelo, Jin Jung, Anne Le. Effects of carcinoma-associated fibroblasts on cancer metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2401.
Abstract 2447: Loss of mitochondrial function and increases in levels of oxidative stress pinpointed in human lung cell cultures exposed to particulate matter from September 11, 2001
Authors: DiLorenzo Ann Marie , Seder Lara , Madrid Rebekah , Tobias Eric , Ortiz Jonathan , Gatson Vern
Published: August 2018

Not only did more than 2,753 individuals pass away the morning of September 11,2001, but there have been individuals suffering from mental and physical illness ever since. Thousands of individuals suffered from breathing complications, asthma, and lung cancer. Clinical trials on cancer patients, including a patient who was exposed to the World Trade Center Dust (WTCD), have seen a decrease in the progression of tumor growth with use of oral nicotinamide adenine dinucleotide (NADH). NADH is naturally occurring within a cell's mitochondria and aids in the production of adenosine triphosphate (ATP), the energy of the cell. NADH is an anti-aging, energy enhancing, substance that also reduces fatigue and successfully treats degenerative medical conditions. NADH is becoming a possible treatment for tumor regression but it is still unknown, on a cellular level, why NADH reacts with these cells to prevent further tumor growth. In this study, the amount of NADH produced by normal MRC-5 lung fibroblast cells will be compared to lung cells exposed to World Trade Center Dust (WTCD), using a Promega NAD/NADH-Glo Assay. The same cells will be evaluated to note the decline in Glutathione to determine the amount Reactive Oxidative Species ( ROS) brought on to lung cells by WTCD. ROS can decrease GSH levels by causing oxidative stress that leads to apoptosis. It is important for the cells to maintain high levels of reduced GSH and low levels of oxidized GSH. PROMEGA GSH Glo Glutathione assay will be used to determine if the toxic, mutagenic and apoptotic effects of WTC dust can be shown as the result of oxidative stress. The cells will be exposed to various concentrations of WTCD, ranging from 25-250 ppm. It is hypothesized that the cells with the highest concentrations of this toxic particulate matter will experience the most accumulation of oxidative stress, resulting in less NAD+, being reduced to NADH, within the mitochondria and simultaneous decrease in protective antioxidants as shown in changes in levels of Glutatione. This should allow correlations to be made on a more precise cellular mechanism between exposure to this WTCD, oxidative stress, and loss of mitochondrial function seen in the diseases of the exposed first responder population.

Citation Format: Ann Marie DiLorenzo, Lara Seder, Rebekah Madrid, Eric Tobias, Jonathan Ortiz, Vern Gatson. Loss of mitochondrial function and increases in levels of oxidative stress pinpointed in human lung cell cultures exposed to particulate matter from September 11, 2001 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2447.
Abstract 2463: Long noncoding RNAs in tumor responses toward lovastatin
Authors: Alanisi Entkhab M. , Wang Jiuhui , Guo Yande , Nie Daotai
Published: August 2018

Background: Lovastatin is one of FDA-approved drugs used to treat hypercholesterolemia to reduce cardiovascular diseases. It is a compound of the statin family derived from type of species of Aspergillus terruses. Lovastatin targets hepatocytes and blocks the cholesterol synthesis pathway by inhibiting the rate-limiting enzyme 3-hydroxy-3-methyl glutaryl CoA reductase (HMG-CoA reductase). This leads to decreased production of cholesterol and many other biochemical end products, including ubiquinone and dolichol. These biochemical end products are important for cancer progression. When lovastatin blocks their production, it leads to increased apoptosis of cancer cells. Therefore, lovastatin has promising anticancer activities but the detailed mechanisms remain poorly understood. Long noncoding RNAs are originally thought as nonfunctional. Emerging evidence suggest that lncRNAs have crucial roles in many biologic functions like apoptosis and cell cycle, translation control, epigenetic regulation and splicing regulation. In this study, we set out to identify and determine Lnc-RNAs that can regulate tumor cell responses towards lovastatin treatment.

Methods: PC3, Heya8, HCT116 cells were obtained from ATCC. HCT116 p53-/- cells were gifts from Dr. Yin-Yuan Mo. The cytotoxic effects of lovastatin on those cell lines were evaluated by several approaches like MTS assays and morphologic changes. The qPCR array was used to identify lncRNAs whose levels are changed by lovastatin treatment. The regulation of lncRNA by lovastatin was further confirmed in multiple cell lines.

Results and Conclusion: Lovastatin treatment had cytotoxic effects in multiple cell lines we evaluated. Through profiling array, we have identified NEAT1, MALAT1, and RMST as a responder to lovastatin. The lncRNAs identified had a significant increase in the expression compared to the nontreated cells in a dose- and time-dependent manner. The RMST mRNA expression in the HCT116 p53-/- cell line was about 25-fold more than its level in the HCT116 wild-type cells after treatment of 20 μM lovastatin for 48 hours, suggesting a role of tumor suppressor p53 gene in regulating the RMST expression. We knocked out RMST using CRISPR-Cas9 approach and found that RMST is an important mediator for lovastatin to downregulate mTOR activities and reduce colony formation. Taken together, our data suggest that lncRNAs can be stimulated by lovastatin to elicit cellular responses.

Citation Format: Entkhab M. Alanisi, Jiuhui Wang, Yande Guo, Daotai Nie. Long noncoding RNAs in tumor responses toward lovastatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2463.
Abstract 2467: Role of PCA3 in prostate cancer cell proliferation and progression
Authors: Russel Bethanie , Wang Jiuhui , Zhang Yuanqin , Nie Daotai
Published: August 2018

Background

PCA3 is a long non-coding RNA used as an important biomarker in early prostate-cancer diagnosis but its biological functions are not understood. The aim of the study is to investigate the potential role of PCA3 in prostate cancer (PCa) initiation, growth and progression.

Methods

Human genomic DNA was used as templates for cloning of PCA3 gene. Primers were designed and polymerase chain reactions were performed. The 3 exons of PCA3 were cloned and confirmed by sequencing. To determine the impact of PCA3 and its individual exon in PCa cell growth and survival, we over-expressed them respectively in PC-3 and DU145 cell lines via transduction with lentiviral vector. qPCR was performed to examine the expression level of PCA3. To investigate the effects of PCA3 on prostate cancer cells, cell proliferation assay was performed by using trepan blue staining. Colony formation assays were performed to measure the clonogenic capacity of the cells after PCA3 overexpression and treatments with docetaxel.

Results and conclusions

Sequencing of PCA3 gene amplified from human tumor cells did not find mutations when compared to RefSeq. We overexpressed PCA3 in prostate cancer cells and found that increased PCA3 expression did not have significant impacts on DU145 or PC-3 cell proliferation. However, PCA3 exon 3 did increase the clonogenic capacity of DU145 cells when compared to the controls. DU145 cells with increased PCA3 exon 3 also presented increased resistance to docetaxel. No significance was observed in clonogenic capacity in transduced PC-3 cells, but PCA3 increased the motility of PC-3 cells. Our studies suggest that PCA3 can modulate prostate cancer cell clonogenic capacity, docetaxel resistance, and motility. Further studies are ongoing to determine the mechanisms involved.

Citation Format: Bethanie Russel, Jiuhui Wang, Yuanqin Zhang, Daotai Nie. Role of PCA3 in prostate cancer cell proliferation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2467.
Abstract 2468: Increased expression of snoRNA in the tumor vasculature
Authors: Sample Klarke M. , Nie Yingjie , Bicknell Roy
Published: August 2018

The tumor microenvironment has long been known to cause the differential expression of many genes within the endothelial cells that comprise the tumor vasculature. Yet, the differential expression of short nuclear RNA (snoRNA) has not previously been observed within tumor associated endothelial cells. A differential gene expression analysis was conducted on total RNAseq data derived from primary endothelial cell isolates and validated by qPCR. By these means, SNORD75, SNORD76, SCARNA7 and SNORA81 were demonstrated to be expressed at a higher level in liver, lung and colon associated tumor endothelial cells when compared to patient matched endothelial cells from healthy adjacent tissue. This data suggests that these four snoRNA molecules could be contributing to pathological angiogenesis and therefore tumor growth. Further work is currently being conducted to investigate this possibility.

Citation Format: Klarke M. Sample, Yingjie Nie, Roy Bicknell. Increased expression of snoRNA in the tumor vasculature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2468.
Abstract 2497: Inhibitors of RNA Pol I and an aminoacyl-tRNA synthetase result in MYC and MYCN downregulation in MYC family protein-driven neuroblastoma cells
Authors: Ikegaki Naohiko , Zeki Jasmine , Chiu Bill , Shimada Hiroyuki
Published: August 2018

We have previously reported histologic variants among unfavorable histology neuroblastomas (NB), having large cell appearance with vesicular nuclei and or prominent nucleolar (PN) formation, indicating hyperactive rRNA synthesis and protein translation. These features appear to be associated with dismal outcome and high-level MYC family protein expression. PN formation could provide critical prognostic and risk-stratification information for NB. At the same time, this observation could open up an opportunity for innovative therapy for aggressive NB. To assess this possibility, we examined the effect of two potent and specific small-molecule inhibitors of RNA Pol I activity and an aminoacyl tRNA synthetase (CX-5461 and Halofuginone, respectively) on growth and MYC family protein expression in NB cell lines. These small-molecule inhibitors inhibited growth of NB cell lines at low to submicromolar concentrations in vitro in 48 hours. Moreover, the inhibitors destabilized MYC and MYCN proteins in NB cells. Interestingly, halofuginone showed a rapid effect on the stability of MYC family proteins at 500 nM-1 uM (<3 hours). In contrast, CX-5461 at 1 uM was less effective at the 3-hour time. At the concentration of 100 nM-250 nM for halofuginone and 1 uM for CX-5461 for up to 48 hours of the drug treatments, MYC and MYCN expression were both downregulated. We further examined the preclinical efficacy of halofuginone in orthotopic Kelly human NB xenografts in mice using a silk film as the drug delivery device. Halofuginone showed a significant growth-suppressive effect on the xenografts, as indicated by the time periods to reach certain tumor sizes being significantly longer in the halofuginone-treated group than those in the control group. Moreover, histologic examination of the xenografts showed a marked suppression of MYCN protein expression in the halofuginone-treated xenograft at day 17 of the drug treatment. Specific inhibition of hyperactive rRNA synthesis and protein translation was shown to be an effective way to suppress MYC/MYCN protein expression and NB growth. Together, MYC-family protein overexpression and PN formation should be included in new NB risk stratification and considered for potential therapeutic targets.

Citation Format: Naohiko Ikegaki, Jasmine Zeki, Bill Chiu, Hiroyuki Shimada. Inhibitors of RNA Pol I and an aminoacyl-tRNA synthetase result in MYC and MYCN downregulation in MYC family protein-driven neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2497.
Abstract 2508: Effect of ALK-inhibitors in synovial sarcoma (SS) cell line HS-SYII
Authors: Gyotoku Hiroshi
Published: August 2018

Synovial sarcoma (SS) accounts for approximately 8% of all soft tissue sarcomas (STS). The prognosis is poor because of a few effective chemotherapy regimens for STS such as Doxorubicin, Eribulin, and Trabectedin. Pazopanib, a multi-targeted tyrosine kinase inhibitor of PDGFR, VEGFR and c-Kit, is the first molecular targeted drug approved by FDA for STS. Recently, a preclinical efficacy of MET-inhibitor and ALK-inhibitor for a synovial sarcoma cell line was reported. We found that the response to ALK-inhibitors (Alectinib, Crizotinib and Lorlatinib) for SS cell line HS-SY-II different from Aska-SS cell line in a previous report. In addition, a combination of ALK-inhibitor and Pazopanib, previously reported that single-agent is effective in HS-SY-II, potentiates a cytotoxic effect by MTT assay than each single agent. Western blotting revealed that both of ALK-inhibitor single agent and the combination of Pazopanib and ALK-inhibitor suppress the phosphorylation of ERK and AKT, downstream of MAPK and PI3K pathway. Our data suggests that the combination therapy Pazopanib and ALK-inhibitor may be effective to SS patients clinically.

Citation Format: Hiroshi Gyotoku. Effect of ALK-inhibitors in synovial sarcoma (SS) cell line HS-SYII [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2508.