bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1385: Patient-derived xenografts as validation material for NGS assays: Analysis of genetic homogeneity
Authors: Konigshofer Yves , Anekella Bharathi , Garlick Russell
Published: July 2016

Full process controls and validation materials for next generation sequencing (NGS)-based molecular diagnostics in Oncology that mimic a biopsy sample and are renewable and scalable have been elusive. Patient-derived Xenografts (PDXs), which are similar in morphology and somatic mutations to original tumors, may present a solution to this pressing need.

In order to evaluate the abilities of PDXs to serve as full process controls and validation materials, we had PDXs that contained established driver mutations KRAS G12C analyzed by clinical laboratories using a variety of different NGS-based assays. The resulting data was analyzed for genetic homogeneity as well as for the presence of mouse-derived variants.

PDXs were found to be generally compatible with NGS-based assays and similar variant frequencies of driver mutations were reported. High concordance was also observed in variant frequencies of PDXs at passage 5 that were derived from the same tumor but grown in different mice. For example, the observed differences in over 90% of variants and all driver mutations were not statistically significant.

In amplicon-based assays, which represent the majority of current assays that look at mutational hotspots, mouse-derived variants were rare. Typically, variant data was usable without any further processing. In hybrid/capture-based assays, mouse-derived variants were common because of the lower binding stringency used by these assays. Without further processing, this manifested in slightly lowered variant frequencies for driver mutations - where wild-type “reference” sequences are often conserved between species and thus inflate the number of reference sequences - and greatly elevated numbers of total variants. However, by processing the data at the FASTQ stage using in-house developed software, it was possible to effectively remove mouse-derived variants in a manner that would be compatible with patient testing as well.

In conclusion, we show that PDXs are compatible with NGS-based assays and can be used to control the full process from the point where the Pathologist first fixes the tissue. Those containing driver mutations that have been previously characterized in cancer also enable laboratories to control and validate their assays by showing that they can consistently detect these mutations.

Citation Format: Yves Konigshofer, Bharathi Anekella, Russell Garlick. Patient-derived xenografts as validation material for NGS assays: Analysis of genetic homogeneity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1385.
Abstract 1387: Application of target enrichment combined with unique molecular identifiers to determine allelic frequencies of human cancers
Authors: Kraushaar Daniel C. , Bowman Sarah K. , Davis Theodore B. , Emerman Amy B. , Henig Noa , Patel Kruti M. , Russello Salvatore , Hendrickson Cynthia L.
Published: July 2016

Existing target enrichment and library preparation for next-generation sequencing typically utilize PCR amplification steps that introduce substantial bias and sequencing of duplicate reads, which in turn affect the quantification accuracy of somatic allele frequencies. Unique molecular identifiers (UMIs) are random molecular tags that uniquely label each molecule prior to library amplification and aid in the identification of true PCR duplicates and thereby improve the accuracy in estimating the relative molecular copy number of the original sample. Here, we applied a target enrichment method for hybridization-based capture of regions of interest, combined with unique molecule labeling and next-generation sequencing to determine allelic frequencies of select cancer targets. Target enrichment and library preparation can be performed in just under seven hours, and our UMI method is compatible with sample indexing using separate barcodes. To demonstrate the robustness and accuracy of our method, we present the detection and assessment of allelic frequencies of multiple cancer genes across multiple reference standards. The result is a fast and cost-efficient method that enables the determination of even low allelic frequencies with high confidence.

Citation Format: Daniel C. Kraushaar, Sarah K. Bowman, Theodore B. Davis, Amy B. Emerman, Noa Henig, Kruti M. Patel, Salvatore Russello, Cynthia L. Hendrickson. Application of target enrichment combined with unique molecular identifiers to determine allelic frequencies of human cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1387.
Abstract 1391: Gene expression profiling for cancer classification in circulating tumor cells
Authors: Soifer Harris S. , Salunga Ranelle C. , Harris Tristan G. , Ramirez Jose , Gogoi Priyadarshini , Wang Yixin , Sepehri Saedeh , Handique Kalyan , Schnabel Catherine A.
Published: July 2016

Background: Enumeration and molecular characterization of circulating tumor cells (CTCs) offer a non-invasive method for tumor analysis, particularly in cases where the biopsy is difficult to obtain or has been exhausted by conventional diagnostic methods. The 92-gene assay (CancerTYPE ID®, bioTheranostics, Inc.) is a clinically-validated cancer classifier based on the collective expression of 87 informative genes, many of which are involved in lineage commitment and signal transduction. The objective of this study was to determine the feasibility of a blood-based application using differential gene expression.

Methods: Technical feasibility studies for cancer cell enrichment and RNA viability were conducted by purifying cancer cell lines that were spiked into donor blood using a microfluidic chip (Celsee PREP™100 system, Celsee Diagnostics) followed by immunodepletion of leukocytes. Quantitative RT-PCR and immunoflourescence were performed on cells harvested at different steps of the purification workflow. Quantitative RT-PCR on individual genes specific to each lineage compartment was used to estimate cell types and cell numbers that were co-purified by the workflow. Gene expression was evaluated by the 92-gene assay.

Results: An integrated workflow was established that allows for the molecular characterization of cancer cells purified from blood. Cell enrichment on the microfluidic chip followed by purification using immunodepletion resulted in efficient recovery (> 80% of input cells) and viable RNA from cancer cells spiked into whole blood. A key result of this study was the demonstration that the level of cancer-specific gene expression in purified samples was similar to the level of expression from an equivalent number of unspiked cancer cells suggests that significant cell loss does not occur during the purification workflow. A comparison of gene expression profiles from purified and mixed cell populations allowed for the identification of subsets of genes that are differentially expressed in cancer cells compared with leukocytes.

Conclusion: A functional workflow with viable RNA and efficient recovery of cancer cells that are within the technical specifications of the 92-gene assay was established. Findings from this feasibility study are fundamental to the potential for CTC-based cancer classification.

Citation Format: Harris S. Soifer, Ranelle C. Salunga, Tristan G. Harris, Jose Ramirez, Priyadarshini Gogoi, Yixin Wang, Saedeh Sepehri, Kalyan Handique, Catherine A. Schnabel. Gene expression profiling for cancer classification in circulating tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1391.
Abstract 1393: Developing a next-generation noninvasive clinical test for non-small cell lung cancer
Authors: Kasbek Christopher , Song Yang , Quintanilha Danielle , Chen Si , Song QingXuan , Wang Tianjiao , Huang Jun T.
Published: July 2016

Significance: Non-Small Cell Lung Cancer (NSCLC) is responsible for the largest number of cancer-related fatalities, accounting for 22.8% of all cancer deaths in the United States in 2015. Targeted therapy precisely treats mutated cancer pathways and has significantly higher response rates compared to chemotherapy. However, tumor heterogeneity is prevalent in NSCLC patients, and the common practice of detecting mutations using tissue biopsy from a fraction of a single tumor can be less than satisfactory. Failure to detect molecular resistance emerging from heterogenic tumor cell population is a major reason that, even though highly effective, targeted therapy has not contributed greatly to prolong patient survival. We aim to develop a cost-effective, highly sensitive and specific liquid biopsy for advanced stage NSCLC patients that is able to capture a comprehensive tumor profile and monitor tumor response to therapy. Innovation: BEST (Blocker-based Enrichment System for Tumor DNA) blocks primer extension of over 90% of wild type DNA and allows preferential amplification of the mutant allele. BEST NSCLC liquid biopsy is highly sensitive (limit of detection of > = 0.005%), comprehensive (detects 56 mutations in the EGFR, KRAS, BRAF, ALK, and ROS1 oncogenes), and rapid (estimated 4-day turnaround time). BEST is comprised of a multiplex of circulating tumor DNA enrichment reaction and a multiplex plasma cDNA enrichment reaction. Our BEST NSCLC liquid biopsy panel enriches >100 fold for the above driver mutations. The ability to detect a myriad of mutations in heterogenic tumor cell populations allows dynamic and personalized treatment during the course of the disease. Summary: Our primary focus is to offer a test to detect actionable mutations and subsequently monitor tumor dynamics and the rise of molecular resistance. Our liquid biopsy will be also ideal for patients in follow-up visits in conjunction with the typical X-ray or CT image scans. Our test is also suitable for vulnerable patients from whom a solid biopsy cannot be obtained due to illness or inaccessibility.

Citation Format: Christopher Kasbek, Yang Song, Danielle Quintanilha, Si Chen, QingXuan Song, Tianjiao Wang, Jun T. Huang. Developing a next-generation noninvasive clinical test for non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1393.
Abstract 1476: A therapeutic antibody that inhibits CD73 activity by dual mechanisms
Authors: Barnhart Bryan C. , Sega Emanuela , Yamniuk Aaron , Hatcher Sandra , Lei Ming , Ghermazien Haben , Lewin Anne , Wang Xi-Tao , Huang Haichun , Zhang Pingping , Korman Alan
Published: July 2016

CD73 has a central role in dictating the adenosine concentration within the tumor as it is the final step in converting extracellular ATP to adenosine. Thus, substantial reduction of CD73 enzymatic activity has the potential to reduce immunosuppression of effector immune cells within the tumor. We present data describing an anti-human CD73 antibody that suppresses CD73 by two mechanisms: 1. direct inhibition of enzymatic activity upon binding to CD73 and 2. rapid, near-complete internalization of the enzyme. Durable reduction of cell-surface CD73 was observed in multiple tumor cell lines both in vitro and in vivo. The unique properties of this antibody are a result of the use of a human IgG2-IgG1 hybrid antibody with effector function eliminated by specific mutations of the Fc. The IgG2 sequence of this antibody drives superior internalization of CD73 and enhanced CD73 inhibition. Syngeneic tumor models demonstrate that CD73 contributes to resistance to anti-tumor therapy. Combination therapy with PD-1 blockade and a surrogate anti-mouse-CD73 antibody resulted in a better anti-tumor efficacy than either treatment alone. Finally, we demonstrate a novel technique for assessing CD73 enzymatic activity in situ that has potential for clinical application. These data support antibody-based anti-CD73 therapy in cancer and highlight a novel mechanism for inhibition of CD73 enzymatic activity.

Citation Format: Bryan C. Barnhart, Emanuela Sega, Aaron Yamniuk, Sandra Hatcher, Ming Lei, Haben Ghermazien, Anne Lewin, Xi-Tao Wang, Haichun Huang, Pingping Zhang, Alan Korman. A therapeutic antibody that inhibits CD73 activity by dual mechanisms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1476.
Abstract 1477: Targeting immunosuppressive myeloid cells in oral cavity cancer with the PI3Kδ/γ isoform inhibitor duvelisib
Authors: Davis Ruth , Moore Ellen , Clavijo Paul E. , Silvin Chris , Van Waes Carter , Chen Zhong , Allen Clint
Published: July 2016

BACKGROUND: IPI-145 (duvelisib) is an oral inhibitor of the p110δ/γ isoforms of phosphinositide 3-kinase (PI3K) that are differentially expressed in leukocytes. IPI-145 is currently under study in phase II/III trials for the treatment of hematologic malignancies, but has not previously been studied in solid tumors. In this study we sought to determine if p110δ/γ inhibition could suppress solid tumor growth through functional inhibition of myeloid derived suppressor cells (MDSCs) within the tumor microenvironment of syngeneic murine oral cavity (MOC) tumor models of head and neck cancer.

METHODS: In pilot experiments, highly immunogenic MOC1 and poorly immunogenic MOC2 tumor-bearing mice were treated with daily oral doses of 15 mg/kg IPI-145 for 14 days and monitored for tumor progression. Flow cytometry was used to study cellular immune correlates in both the periphery and tumor microenvironment. MDSCs were sorted using FACS and functionally evaluated in a carboxyfluorescein succinimidyl ester (CFSE)-based T-cell proliferation assay.

RESULTS: Selective inhibition of p110δ/γ with low-dose IPI-145 resulted in a trend toward growth suppression of established MOC1 tumors, and had no effect on the growth of poorly immunogenic MOC2. IPI-145 increased infiltration and activation of CD8 T-lymphocytes and NK cells along with increased tumor cell expression of MHC class I and PD-L1 in MOC1 tumor bearing mice. MDSCs sorted from IPI-145 treated MOC1 tissues demonstrated a reduction in their capacity to suppress CD3/28 stimulated T-cell proliferation. MOC2 tumor bearing mice demonstrated no immune correlative changes.

CONCLUSION: Daily oral treatment with low-dose IPI-145 resulted in a trend toward inhibition of primary tumor growth and enhanced immune activation in the tumor microenvironment in an immunogenic murine model of head and neck cancer. This data shows promise for future studies of IPI-145 as a monotherapy at higher doses or as a sensitizer for immunotherapies such as checkpoint inhibitors, and these studies are underway.

This work was supported by the NIDCD Intramural Program ZIA-DC-000087-01

Citation Format: Ruth Davis, Ellen Moore, Paul E. Clavijo, Chris Silvin, Carter Van Waes, Zhong Chen, Clint Allen. Targeting immunosuppressive myeloid cells in oral cavity cancer with the PI3Kδ/γ isoform inhibitor duvelisib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1477.
Abstract 15: Metabolomic profiling of cell death in human lung cancer cells by a novel digitoxin analog
Authors: Kulkarni Yogesh , Azad Neelam , Kaushik Vivek , Yakisich Juan Sebastian , Venkatadri Rajkumar , Wright Clayton , Rojanasakul Yon , O’Doherty George , Iyer Anand Krishnan V
Published: July 2016

Background:

Digitoxin, a cardiac glycoside had shown considerable promise as an anti-cancer therapeutic. However, doses of digitoxin required to inhibit cancer cell proliferation lead to cardiotoxic side effects. We have previously shown that a novel analog of digitoxin, MonoD, activates apoptosis in human lung cancer cells at much lower concentrations as compared to digitoxin. We postulate that treatment with MonoD leads to distinct metabolic signatures on lung cancer cells as compared to digitoxin, which will help delineate the pathways that dictate MonoD action.

Experimental Approach:

NCI-H460 cells from identical passages were used for metabolite analysis using an optimized version of the methanol/water (80:20) protocol for metabolite extraction. The supernatant containing metabolites were suspended in 0.1% formic acid spiked with an internal standard for Q-Exactive analysis. The data was analyzed using SIEVETM 2.0 software with the component extraction algorithm that has been specifically designed for optimal data analysis in untargeted metabolomics experiments. Metabolites that were found to be significantly different between treatment groups were detected via Chemspider/HMDB database search.

Results:

We observed several metabolites along the prostaglandin synthesis pathway which were downregulated in MonoD treated lung cancer cells. Prostaglandins play a key role in the generation of the inflammatory response. Chronic inflammation has been shown to be a causative factor in a variety of cancers. Cyclooxygenase-2 (COX-2) derived prostaglandin has been previously shown to promote tumor growth by binding its receptors and activating signaling pathways which control cell proliferation, migration, apoptosis, and angiogenesis. Using the metabolite profiling data, we posited that COX-2, a key enzyme involved in prostaglandin synthesis, in addition to NFκB, a key transcription factor of proinflammatory signaling pathway are downregulated with MonoD treatment. Immunoblotting confirmed the downregulation of COX-2 and NFκB in MonoD-treated cells.

Conclusion: Using metabolite profiling, we have elucidated an important mode of action for MonoD in lung cancer cells, and shown that MonoD exerts its anti-tumorigenic properties by downregulating key factors involved in inflammatory response.

Citation Format: Yogesh Kulkarni, Neelam Azad, Vivek Kaushik, Juan Sebastian Yakisich, Rajkumar Venkatadri, Clayton Wright, Yon Rojanasakul, George O’Doherty, Anand Krishnan V Iyer. Metabolomic profiling of cell death in human lung cancer cells by a novel digitoxin analog. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 15.
Abstract 1504: Identification of novel kinase fusion transcripts in endometrial cancer
Authors: Tamura Ryo , Yoshihara Kosuke , Suda Kazuaki , Yamawaki Kaoru , Ishiguro Tatsuya , Adachi Sosuke , Enomoto Takayuki
Published: July 2016

The prognosis of patients with advanced stage or recurrent endometrial cancer remains poor and there is at present no effective therapeutic molecular target for this disease. Our aim is to identify therapeutic targetable targets in endometrial cancer. We focused on fusion transcripts whose inhibitors show impressive activity in some tumors and have analyzed RNA-sequencing data and DNA copy number data from 25 endometrial cancer cell lines to identify potential therapeutically targetable fusion transcripts. We have identified 124 high-confidence fusion transcripts, of which 69% are associated with gene amplifications but have not detected recurrent fusion transcripts. We performed Sanger sequencing of RT-PCR amplification products and validated 27 (93.1%) of the 29 fusion transcripts that were randomly selected. As targetable fusion candidates, we extracted three in-frame kinase fusion transcripts that retain a kinase domain (CPQ-PRKDC, CAPZA2-MET, and VGLL4-PRKG1). We detected only CPQ-PRKDC fusion transcript in three of 122 primary endometrial cancer tissues. Cell proliferation of the fusion-positive cell line was inhibited by knocking down the expression of wild-type PRKDC but not by blocking the CPQ-PRKDC fusion transcript expression. Quantitative real-time RT-PCR demonstrated that the expression of the CPQ-PRKDC fusion transcript was significantly lower than that of wild-type PRKDC, corresponding to a low transcript allele fraction of this fusion, based on RNA-sequencing read counts. Western blotting did not show the detectable protein level of the CPQ-PRKDC similar to CAPZA2-MET, and VGLL4-PRKG1. In endometrial cancers, the CPQ-PRKDC fusion transcript may be a passenger aberration related to gene amplification. In conclusion, we have described the landscape of fusion transcripts in endometrial cancer by using RNA-sequencing data. We could not identify therapeutically targetable kinase fusions in a small set of 25 endometrial cancer cell lines. Our findings suggest that the transcript allele fraction is a good predictor for determining bona fide targetable fusion transcripts when applying fusion detection methods that use RNA-sequencing data.

Citation Format: Ryo Tamura, Kosuke Yoshihara, Kazuaki Suda, Kaoru Yamawaki, Tatsuya Ishiguro, Sosuke Adachi, Takayuki Enomoto. Identification of novel kinase fusion transcripts in endometrial cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1504.
Abstract 1528: Selective gene-expression profiling of metastasizing breast tumor cell subpopulations complements the predictive power of Mammaprint Dx and Oncotype Dx
Authors: Karagiannis George S. , Goswami Sumanta , Jones Joan G. , Oktay Maja H. , Condeelis John S.
Published: July 2016

Gene-expression profiling has yielded prognostic tests, such as MammaPrint and Oncotype Dx, which facilitate clinical decision-making in breast cancer patients. These tests are based on signatures constructed from whole tumor tissue, including tumor as well as stromal cells. Since metastatic tumor cells are not uniquely identified in these signatures, these tests may have reduced clinical specificity and sensitivity in predicting risk of metastasis. In contrast, we captured breast cancer cells in the act of migration/dissemination using an in vivo invasion assay and expression profiled these cells to create a distinct signature named the human invasion signature (HIS). A comparison between HIS and MammaPrint in the NKI295 cohort demonstrated that both signatures perform comparably in selecting a group of patients with significantly poorer outcomes. However, when compared to MammaPrint, the initial slope of the high-risk patients identified by the HIS is significantly steeper, suggesting that the HIS may identify patients at higher risk of early distant metastatic recurrence. Therefore HIS carries prognostic information beyond that captured by MammaPrint. Additionally, a more thorough examination of candidate genes present in HIS yielded two predictive tissue-based biomarkers: TMEM score which measures the density of cancer cell intravasation sites, and MenaCalc which measures the expression of invasive isoforms of the actin-regulatory protein Mena (MenaCalc = PanMena-Mena11a), thereby identifying tumor cells that disseminate. Both, TMEM density and MenaCalc independently predict the development of distant metastases in breast cancer patients. Here, we show that in a small patient cohort (n = 58), there is no correlation between TMEM density and the Oncotype recurrence score (RS). In particular, a subgroup of patients had tumors with high-Oncotype DX and low TMEM score while another subgroup had low Oncotype DX and high TMEM score. These findings indicate that TMEM and Oncotype Dx address different tumor biology and could be used in a complementary fashion for more accurate patient stratification, as well as better selection of systemic therapies to avoid over- and under-treatment.

Citation Format: George S. Karagiannis, Sumanta Goswami, Joan G. Jones, Maja H. Oktay, John S. Condeelis. Selective gene-expression profiling of metastasizing breast tumor cell subpopulations complements the predictive power of Mammaprint Dx and Oncotype Dx. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1528.
Abstract 1536: In silico discovery of target genes and pathways associated with breast cancer brain and bone metastases
Authors: Tang Su-Ni , Jiang Peixin , Liu Xinli
Published: July 2016

Metastasis is the primary cause of death from breast cancer. Bone and brain are the most frequent sites for breast cancer metastasis. The aim of this study was to explore candidate genes associated with breast cancer bone and brain metastases. We compared the gene expression profiles of bone and brain metastases with primary tumors by compiling 55 microarray data sets from Gene Expression Omnibus (GEO: GSE12763, primary tumor n = 30; GSE14017, bone metastasis n = 10, brain metastasis n = 15). A total of 160 differentially expressed genes were screened with a two-fold change, among which 109 genes were up-regulated and 51 genes were down-regulated in bone metastasis. Pathway analysis showed these differentially expressed genes were significantly related to bone development and cell adhesion (p<0.01). Similarly, a total of 150 differentially expressed genes were identified in brain metastasis, 36 genes were up-regulated and 114 genes were down-regulated, which were enriched in immune response (p<0.01). Moreover, protein-protein interaction analysis revealed highly connected hub genes that are expected to play an important role in brain and bone metastases. These hub genes were FOS, EGR1, CTNNB1, DCUN1D1, HIST1H4E, VIM, UBD, EGFR in bone metastasis, and HIST1H4E, DCUN1D1, CDC42, ESR1 in brain metastasis. Interestingly, two genes, DCUN1D1 and HIST1H4E were both up-regulated in bone and brain metastases. It is known that DCUN1D1 could induce extracellular matrix invasion while HIST1H4E played a critical role in regulating chromosomal accessibility and stability. These findings reveal potential biomarkers and therapeutic targets of breast cancer bone and brain metastases, which provide a molecular basis for our future in vitro and in vivo studies.

Citation Format: Su-Ni Tang, Peixin Jiang, Xinli Liu. In silico discovery of target genes and pathways associated with breast cancer brain and bone metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1536.
Abstract 1540: Midkine-raised oral cancer growth, migration and invasion is required for RBPJk modulation
Authors: Chiu Tai-Jan , Chen Yi-Ching , Chen Chang-Han
Published: July 2016

Oral squamous cell carcinoma (OSCC) represents 95% of all forms of head and neck cancer, and its incidence has increased by 50% during the past decade. Despite the nowadays available therapeutic managements, such as surgery, radiotherapy, chemotherapy or in combination, the five-year survival rate is still improved. The current view is to present that the accumulations of genetic and epigenetic alterations in OSCC lead to the progression of cancer. Our previous study indicated that midkine, heparin-binding growth factor, was overexpressed in tumor tissues of head and neck cancer patients and its expression also correlated with poor 5-year survival. In this study, we tried to explore the biological functions and mechanisms of midkine in vitro and in vivo. Established gain-of-function of midkine in two oral cancer cell lines not only promoted cell growth by MTT, soft agar and BrdU assays, but also induced tumor growth in xenograft model. Midkine-overexpressing cells induced cell migration and invasion by wound healing and Transwell assays. Conversely, cell growth, migration and invasion were also inhibited; while endogenous midkine was abolished by midkine-mediated siRNA in oral cancer cells. By using bioinformatics, Q-RT-PCR, Western blotting, promoter assay and ChIP assay, we identified that RBP-Jk, a transcriptional factor, could bind to the promoter regions of midkine and to induce midkine expression in oral cancer cells. In the specimens of oral cancer patients, the expressions of midkine and RBP-Jk had a positive correlation by immunohistochemistry. Taken together, these results demonstrated that RBP-Jk regulates midkine expression and involves in midkine-elicited tumor formation in oral cancer.

Citation Format: Tai-Jan Chiu, Yi-Ching Chen, Chang-Han Chen. Midkine-raised oral cancer growth, migration and invasion is required for RBPJk modulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1540.
Abstract 1604: TMPRSS4 induces invasion and proliferation of prostate cancer cells through induction of Slug and cyclin D1
Authors: Lee Yunhee , Kim Semi
Published: July 2016

TMPRSS4 is a novel type II transmembrane serine protease found at the cell surface that is highly expressed in pancreatic, colon, and other cancer tissues. Previously, we demonstrated that TMPRSS4 mediates tumor cell invasion, migration, and metastasis. We also found that TMPRSS4 activates the transcription factor activating protein-1 (AP-1) to induce cancer cell invasion. Here, we explored TMPRSS4-mediated cellular functions and the underlying mechanisms. TMPRSS4 induced Slug, an epithelial-mesenchymal transition (EMT)-inducing transcription factor, and cyclin D1 through activation of AP-1, composed of c-Jun and activating transcription factor (ATF)-2, which resulted in enhanced invasion and proliferation of PC3 prostate cancer cells. In PC3 cells, not only c-Jun but also Slug was required for TMPRSS4-mediated proliferation and invasion. Interestingly, Slug induced phosphorylation of c-Jun and ATF-2 to activate AP-1, establishing a positive feedback loop between Slug and AP-1, and thus induced cyclin D1, leading to enhanced proliferation. Using data from The Cancer Genome Atlas, we found that Slug expression positively correlated with that of c-Jun and cyclin D1 in human prostate cancers. This study demonstrates that TMPRSS4 modulates both invasion and proliferation via Slug and cyclin D1, which is a previously unrecognized pathway that may regulate metastasis and cancer progression.

Citation Format: Yunhee Lee, Semi Kim. TMPRSS4 induces invasion and proliferation of prostate cancer cells through induction of Slug and cyclin D1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1604.
Abstract 1617: RUNX3 inhibits metastasis and angiogenesis in colorectal cancer
Authors: Kim Bo Ram , Kim Jung Lim , Na Yoo Jin , Park Seong Hye , Lee Sun Il , Kang Sanghee , Joung Sung Yup , Lee Suk Young , Kim Hong-Jun , Lee Dae-Hee , Min Byung-Wook , Oh Sang Cheul
Published: July 2016

Recent studies proved that an inactivation of RUNX3 (runt-related transcription factor-3) expression is highly associated with lymph node metastasis and poor prognosis in various cancer types. However, the mechanism of RUNX3-mediated suppression of tumor metastasis remains unclear. Herein, we aimed to clarify the effect of RUNX3 on metastasis and angiogenesis in colorectal cancer (CRC). First, we found that the reduction of expression of RUNX3 in CRC tissues when compared with tumor adjacent normal colon tissues, as indicated by reduced RUNX3 staining, was significantly correlated with TNM stage. Second, we demonstrated that RUNX3 overexpression inhibited CRC cell migration and invasion resulting from the elevated upregulation of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) expression. In contrast, the knockdown of RUNX3 reduced the inhibition of migration and invasion of CRC cells. Last, Vascular endothelial growth factor(VEGF) is an inducer of angiogenesis and lymphangiogenesis. We found that restoration of RUNX3 decreased vascular endothelial growth factor (VEGF) secretion and suppressed endothelial cell growth and tube formation in CRC cells. Taken together, our data demonstrated that RUNX3 may provide insights into the development of RUNX3 for CRC metastasis diagnostics and therapeutics.

Citation Format: Bo Ram Kim, Jung Lim Kim, Yoo Jin Na, Seong Hye Park, Sun Il Lee, Sanghee Kang, Sung Yup Joung, Suk Young Lee, Hong-Jun Kim, Dae-Hee Lee, Byung-Wook Min, Sang Cheul Oh. RUNX3 inhibits metastasis and angiogenesis in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1617.
Abstract 1626: Krüppel-like factor 8 activates the transcription of C-X-C cytokine receptor type 4 to promote breast cancer cell invasion, transendothelial migration and metastasis
Authors: Mukherjee Debarati , Lu Heng , Yu Lin , Lahiri Satadru K. , Li Tianshu , Zhao Jihe
Published: July 2016

Krüppel-like factor 8 (KLF8) has been strongly implicated in breast cancer metastasis. However, the underlying mechanisms remain largely unknown. Here we report a novel signaling from KLF8 to C-X-C cytokine receptor type 4 (CXCR4) in breast cancer. Overexpression of KLF8 in MCF-10A cells induced CXCR4 expression at both mRNA and protein levels, as determined by quantitative real-time PCR and immunoblotting. This induction was well correlated with increased Boyden chamber migration, matrigel invasion and transendothelial migration (TEM) of the cells towards the ligand CXCL12. On the other hand, knockdown of KLF8 in MDA-MB-231 cells reduced CXCR4 expression associated with decreased cell migration, invasion and TEM towards CXCL12. Histological and database mining analyses of independent cohorts of patient tissue microarrays revealed a correlation of aberrant co-elevation of KLF8 and CXCR4 with metastatic potential. Promoter analysis indicated that KLF8 directly binds and activates the human CXCR4 gene promoter. Interestingly, a CXCR4-dependent activation of focal adhesion kinase (FAK), a known upregulator of KLF8, was highly induced by CXCL12 treatment in KLF8-overexpressing, but not KLF8 deficient cells. This activation of FAK in turns induced a further increase in KLF8 expression. Xenograft studies showed that overexpression of CXCR4, but not a dominant-negative mutant of it, in the MDA-MB-231 cells prevented the invasive growth of primary tumor and lung metastasis from inhibition by knockdown of KLF8. These results collectively suggest a critical role for a previously unidentified feed-forward signaling wheel made of KLF8, CXCR4 and FAK in promoting breast cancer metastasis and shed new light on potentially more effective anti-cancer strategies.

Citation Format: Debarati Mukherjee, Heng Lu, Lin Yu, Satadru K. Lahiri, Tianshu Li, Jihe Zhao. Krüppel-like factor 8 activates the transcription of C-X-C cytokine receptor type 4 to promote breast cancer cell invasion, transendothelial migration and metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1626.
Abstract 1634: The RALA pathway drives invasion and metastasis of soft tissue sarcomas by regulating MMP14 localization and activity
Authors: Sizemore Steven T. , Xia Fen , Chakravarti Arnab
Published: July 2016

Soft tissue sarcomas (STS) are a heterogeneous group of tumors originating from mesodermal tissues such as skeletal muscle, adipose, and fibrous tissue. The propensity of STS to metastasize, particularly to the lung which occurs in approximately 20% of all cases, is the major source of mortality associated with this disease. Currently, themolecular mechanisms that drive STS metastasis are poorly understood greatly limiting our ability to determine which STS will metastasize and impeding the development of targeted therapies for the treatment of STS metastases. In this study we utilized analysis of publicly available gene expression data to identify loss of PPP2R1B expression as a strong prognosticator of STS metastasis. In vitro analyses using SW872 liposarcoma (LPS) and HT1080 fibrosarcoma cell lines determined that PPP2R1B plays an essential role in the dephosphorylation and inactivation of RALA, a major downstream effector of the Ras pathway. RALA was found to be essential for invasion of STS cells in modified Boyden chamber assays as knockdown of RALA by either siRNA or shRNA significantly decreased invasion through matrigel. Analysis of publicly available gene expression data revealed that RALA expression correlated with more aggressive LPS subtypes and was prognostic of LPS metastasis. Furthermore, we identify the transmembrane matrix metalloprotease MMP14 (MT1-MMP) as a key downstream effector of RALA mediated invasion in STS. Stable knockdown of RALA in SW872 and HT1080 cells resulted in significant mislocalization of MMP14 and reduced MMP14 enzymatic activity. MMP14 expression was also significantly prognostic of metastasis in a publically available LPS cohort. Finally, we demonstrate that a three gene signature comprised of PPP2R1B, RALA, and MMP14 can retrospectively stratify LPS patients into groups of low, moderate, or high risk for metastatic recurrence. To the best of our knowledge this is the first demonstration of a vital role for PPP2R1A and RALA in STS invasion and metastasis and these data demonstrate a novel role for RALA in the localization and activity of MMP14. In conclusion, we have identified the PPP2R1B-RALA-MMP14 axis as driver of STS metastasis with potential clinical value as both a prognostic marker and therapeutic target.

Citation Format: Steven T. Sizemore, Fen Xia, Arnab Chakravarti. The RALA pathway drives invasion and metastasis of soft tissue sarcomas by regulating MMP14 localization and activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1634.
Abstract 1636: The role of the ROR receptors in ovarian cancer progression and chemoresistance
Authors: Henry Claire E. , Llamosas Estelle , Hacker Neville F. , Heinzelmann-Schwarz Viola , Ford Caroline E.
Published: July 2016

BACKGROUND: Despite recent advances in our understanding of the underlying genetic profile of distinct subtypes of ovarian cancer, overall survival remains poor. This is partly due to the absence of available targeted therapies, widespread metastatic disease at diagnosis, and the development of resistance to chemotherapy in the majority of patients. Patients with a more mesenchymal gene signature have a shorter overall survival, and are more likely to recur and develop chemoresistance. These patients also have upregulated Wnt signalling, a key developmental pathway associated with metastasis, epithelial to mesenchymal transition (EMT) and chemoresistance.

AIM: To investigate the role of the novel Wnt receptors ROR1 and ROR2 in ovarian cancer progression and chemoresistance, to determine their feasibility as therapeutic targets.

METHOD: Expression of ROR1 and ROR2 was measured in patient tumour (primary and metastatic) samples and ascites fluid taken at multiple time points during disease progression. In addition, the A2780 and cisplatin resistant A2780-cis cell lines were used as a model of ovarian cancer chemoresistance. Profiling of EMT and Wnt genes was undertaken alongside cell proliferation, adhesion, migration, invasion and viability assays after modulation of ROR1 and ROR2.

RESULTS: Expression of ROR1 and ROR2 is increased in ovarian cancer patients compared to benign fallopian tube and ovarian surface epithelium. High ROR1 expression is associated with type I epithelial ovarian cancer, and high ROR2 expression with aggressive type II epithelial ovarian cancer. Expression of each receptor fluctuates in paired metastatic samples compared to primary tumours, depending on the site of metastasis and recurrence time. Both receptors are increased in a model of cisplatin resistant ovarian cancer, and when silenced, cell migration and invasion is significantly inhibited. Silencing ROR1 and ROR2 also sensitizes resistant cells to cisplatin, and is associated with a shift away from a mesenchymal phenotype.

CONCLUSION: Upregulation of ROR1 and ROR2 drives migration, invasion and chemoresistance through regulating EMT via β-catenin independent Wnt signalling. As cell surface regulators of β-catenin independent Wnt signalling, the ROR receptors represent potential therapeutic targets for chemoresistant cancers.

Citation Format: Claire E. Henry, Estelle Llamosas, Neville F. Hacker, Viola Heinzelmann-Schwarz, Caroline E. Ford. The role of the ROR receptors in ovarian cancer progression and chemoresistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1636.
Abstract 1644: NF-κB inhibitor DMAPT blocks non-homologous end-joining repair of radiation-induced DSBs in NSCLC
Authors: Deraska Peter V. , O’Leary Colin , Lazaro Jean-Bernard , Sweeney Christopher J. , D’Andrea Alan D. , Kozono David
Published: July 2016

Background: Despite optimal multimodality therapy, non-small cell lung carcinoma (NSCLC) remains the leading cause of cancer-related death in the United States. A common limitation is our inability to provide sufficient radiotherapy (RT) to eradicate tumor due to risk of toxicities in surrounding tissues. There is thus an unmet need for radiosensitizers that can improve the therapeutic index. Dimethylaminoparthenolide (DMAPT), an orally bioavailable small molecule NF-κB inhibitor, inhibits repair of ionizing radiation (IR)-induced DNA double strand breaks (DSBs) and increases control of subcutaneous mouse NSCLC xenografts. While our prior work focused on inhibition of homologous recombination as a mechanism for radiosensitization, we sought to characterize the effect of DMAPT on a second major DSB repair pathway, non-homologous end-joining (NHEJ).

Methods: NHEJ was assessed in NSCLC lines using the pEJ reporter and flow cytometry. The NF-κB super repressor (IκBαS32A,S36A) was used as a control. Immunofluorescence and Western blotting were used to assess NHEJ biomarkers including 53BP1, DNA-PKS2056, Ku70/80 and XRCC4. Cell fractionation was performed to assess Ku chromatin binding. Quantitative RT-PCR was performed to assess gene transcription. Ku complexes were purified to identify binding partners.

Results: NSCLC cells treated with IR-sensitizing doses of DMAPT (5-15 μM) or the NF-κB super repressor showed significant decreases in NHEJ. DMAPT increased the persistence of 53BP1 foci, indicating a failure to complete NHEJ. Regardless of exogenous DNA damage, there was reduced Ku70 chromatin binding following DMAPT treatment. DMAPT-treated cells produced fewer distinct IR-induced DNA-PKS2056 foci. Further, there was decreased IR-induced XRCC4 chromatin recruitment, suggesting that repair was impaired prior to ligation. There was no change in Ku70/80 transcription following DMAPT and/or IR. However, Western blotting of purified Ku complexes showed that DMAPT treatment decreased Ku association with RNA binding partners including RPL19. We also observed decreased recruitment of DNA-PKcs to the Ku complex, suggesting decreased Ku affinity for DSBs.

Conclusions: These results indicate that DMAPT radiosensitizes NSCLC by perturbing the binding affinity of Ku to RNA, DNA and its complex binding partners, thus blocking NHEJ. Further mechanistic investigation and analysis of NHEJ biomarkers in vivo is needed to identify the precise mechanism.

Citation Format: Peter V. Deraska, Colin O’Leary, Jean-Bernard Lazaro, Christopher J. Sweeney, Alan D. D’Andrea, David Kozono. NF-κB inhibitor DMAPT blocks non-homologous end-joining repair of radiation-induced DSBs in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1644.
Abstract 1657: Mapping mechanisms of radiotherapy resistance in prostate cancer
Authors: Bruens Serena T. , Milanese Chiara , Verkaik Nicole , Mastroberardino Pier , Gyenis Akos , Chang Jiang , Derks Kasper , Wiemer Erik , van Gent Dik , van Weerden Wytske , Jenster Guido , Hoeijmakers Jan , Pothof Joris
Published: July 2016

Cancer cells that acquire radio-resistance are major problem for treatment outcome. We aimed at identifying mechanisms and networks that control ionizing radiation (IR)-resistance in prostate cancer cells. To this end, we applied to cancer cell lines an identical radiotherapy regiment as performed in the clinic, i.e. a total dose of 78 Gy in steps of 2 Gy per day for 5 consecutive days followed by 2 days rest. We observed that radio-resistance occurs early during treatment as seen in colony survival assays in which 100% of cells that can form colonies are resistant up to 8 Gy. Remarkably, a two-week period of culturing without radiotherapy completely sensitized these resistant cancer cell lines, indicating that resistance is not acquired by permanent mutations or chromosomal rearrangements. Currently, we are characterizing these extreme resistant cancer cell cultures by comparing these to their sensitive parental cancer cell lines and the two-week re-sensitized cancer cell lines. We observe changes in DNA repair pathway utilization, DNA damage checkpoint activation and energy metabolism parameters, but not in PI3-kinase signalling and cancer stem cell markers. We also performed genome-wide gene expression analysis by next generation sequencing and isolated gene signatures that correlate with resistance. Based on our results we applied inhibitors to sensitize these IR-resistant cells. Notably, application of single inhibitors did not lead to sensitivity. Combinations of small molecule inhibitors however, were able to sensitize these cancer cell lines, indicating that IR-resistance is a multi-factorial process. In summary, mapping gene networks and mechanisms will uncover new pathways associated with IR-resistance.

Citation Format: Serena T. Bruens, Chiara Milanese, Nicole Verkaik, Pier Mastroberardino, Akos Gyenis, Jiang Chang, Kasper Derks, Erik Wiemer, Dik van Gent, Wytske van Weerden, Guido Jenster, Jan Hoeijmakers, Joris Pothof. Mapping mechanisms of radiotherapy resistance in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1657.