bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5399: Investigating phenotypic plasticity in breast cancer with high-throughput nanogrid single-nucleus RNA sequencing
Authors: Gao Ruli , Kim Charissa , Sei Emi , Yang Jie , Chan Leo , Srinivasan Maithreyan , Zhang Hong , Meric-Bernstam Funda , Navin Nicholas E.
Published: July 2017

Single-cell RNA sequencing (RNA-seq) is a powerful tool for investigating rare tumor subpopulations and resolving intra-tumor heterogeneity, but is low throughput, expensive, and requires fresh tissue samples. To address these limitations, we developed a 5’ high-throughput single-nucleus RNA sequencing (SNRS) approach that uses nanogrid technology to perform single-cell imaging and sequencing of 500-2500 nuclei in parallel. The automated image scanning procedure allowed us to exclude doublets and select live cells with DAPI/PI staining. This approach allows the transcriptomic profiling of frozen tissue samples, in which the cytoplasmic membrane is ruptured in cells, but leaves the nuclear membrane intact. We validated SNRS in a breast cancer cell line (SK-BR-3) and compared the transcriptomes of 500 nuclei to 500 whole cells, which revealed a high concordance in the number of genes expressed as well as their expression levels. We also performed bulk RNA-seq of isolated nuclear and cellular fractions from 5 breast cancer cell lines, which showed a high concordance in genes and expression levels. Differentially expressed genes in the nucleus mainly included lincRNAs, pseudogenes and mitochondria genes, but did not affect most cancer genes and pathway analysis. We further applied SNRS to sequence 500 nuclei from a triple-negative breast cancer patient and identified diverse phenotypes in tumor cells, including variation in cell proliferation, migration, invasion, and epithelial-to-mesenchymal transition. These studies demonstrated the technical feasibility of using a nanogrid platform to perform high-throughput single-cell RNA sequencing and showed that nuclei from cell lines and tumors can be used to study signaling pathways and gene networks that play an important role in tumor progression.

Citation Format: Ruli Gao, Charissa Kim, Emi Sei, Jie Yang, Leo Chan, Maithreyan Srinivasan, Hong Zhang, Funda Meric-Bernstam, Nicholas E. Navin. Investigating phenotypic plasticity in breast cancer with high-throughput nanogrid single-nucleus RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5399. doi:10.1158/1538-7445.AM2017-5399
Abstract 540: Deep learning to determine breast cancer estrogen receptor status from nuclear morphometric features in H&E images
Authors: Rawat Rishi R. , Ruderman Daniel , Agus David B. , Macklin Paul
Published: July 2017

We hypothesized that the spatial arrangement of nuclei in a hematoxylin and eosin (H&E) stained image contains a vast quantity of latent information that is currently underutilized. We tested this hypothesis on the classification of breast cancer biopsies as estrogen receptor (ER) positive or negative. While determining ER status typically requires immunohistochemistry (IHC) staining, we aimed to recapitulate this step by predicting ER status directly from H&E stained slides. By training a deep neural network on nuclear-morphometric features, we demonstrate the power of machine learning to provide insights about cancer biology, and aim to scale precision oncology for the developing world.

We constructed a learning pipeline that segments nuclei from H&E stained slides scanned into digital images, extracts shape and orientation descriptors, and uses a deep neural network to learn spatial features to predict ER status. After training the pipeline on 57 tissue cores of invasive ductal carcinoma (IDC), we were able to predict ER status in patients with IDC in a statistically significant manner (AUC=0.72, 95%CI=0.55-0.88, n=56) and ductal carcinoma in situ (AUC=0.82, 95%CI=0.74-0.89, n=106).

To our knowledge, this is the first work to show that nuclear morphometric information can be used to predict the molecular status of a breast cancer tumor. An initial analysis of the features learned by the deep neural network provides a glimpse into the mechanistic differences between ER+ and ER- cancers: suggesting the importance of spatial heterogeneity in distinguishing between ER+ and ER-.

We anticipate that further analysis will provide deeper insights about the morphological differences between ER+ and ER- cancers, and that this type of analysis can be applied to other markers and cancers. Moreover, an optimized version of this pipeline may serve patients in the developing world by providing a means to predict marker status directly from H&E images.

Citation Format: Rishi R. Rawat, Daniel Ruderman, David B. Agus, Paul Macklin. Deep learning to determine breast cancer estrogen receptor status from nuclear morphometric features in H&E images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 540. doi:10.1158/1538-7445.AM2017-540
Abstract 5400: Accurate identification of single nucleotide variants in whole genome amplified single cells
Authors: Dong Xiao , Zhang Lei , Milholland Brandon , Lee Moonsook , Maslov Alexander Y. , Wang Tao , Vijg Jan
Published: July 2017

Single cell sequencing for analyzing DNA mutations across the genome in somatic tissues is critically important for studying development, cancer and aging. However, current procedures are prone to artifacts and to date a reliable protocol for single-cell somatic mutation analysis remains to be developed. Here we address the two largest sources of artifacts, i.e., DNA denaturation-related cytosine deamination and allelic bias-driven whole genome amplification errors. We first reconfigured multiple displacement amplification (MDA) into an efficient protocol for whole genome amplification of single cells without cytosine deamination artifacts, i.e., Single Cell MDA (SCMDA). We then developed a new single-cell SNV caller (SCcaller) that distinguishes real somatic mutations and amplification errors by utilizing a SNP-based localized estimate of allelic amplification bias. The procedure was validated by comparing SCMDA-amplified single cells with unamplified clones derived from single cells from the same population. Together with SCcaller, SCMDA provides a firm foundation for analyzing cellular heterogeneity in somatic mutational landscapes in tumor and surrounding, normal tissues.

Citation Format: Xiao Dong, Lei Zhang, Brandon Milholland, Moonsook Lee, Alexander Y. Maslov, Tao Wang, Jan Vijg. Accurate identification of single nucleotide variants in whole genome amplified single cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5400. doi:10.1158/1538-7445.AM2017-5400
Abstract 5408: RNA fusion detection in thyroid nodules with a targeted RNA sequencing panel
Authors: Tian Song , Baird Michelle , Reinecke Frank , Deng Jixin , Ranjbar Mohammad Nezami , Samara Raed , Lader Eric
Published: July 2017

Thyroid nodules are quite common and detected in as high as 67% of the population. Only a small portion of thyroid nodules are malignant thyroid cancers. Advances in understanding the molecular pathology of thyroid cancer are leading to the development of better biomarkers and novel targeted therapies. The tumorigenic role of RNA fusions has been reported in many forms of cancer. Fusions can result from structural rearrangements like translocations and deletions, transcription read-through of neighboring genes, or the trans- and cis-splicing of pre-mRNAs. Many fusions act as key drivers for cancer development. Detection of fusion RNA is a challenge with traditional technologies like FISH and qPCR, as they are laborious, low throughput, and do not provide detailed information about the fusion. Here we explored the possible use of a tumor fusion-targeted RNA-based panel (QIAseq targeted RNAscan panel, which is targeting 223 fusion gene pairs, 576 unique fusions) as a tool to study fusions in thyroid nodules. The presence of these fusions were surveyed in RNA samples from 10 thyroid cancer (PTC and FVPTC) samples and 19 non-cancer control samples (NH and FA) using this oncology targeted panel and sequenced on the Illumina NextSeq. PAX8-PPARG, a fusion gene with a well-established link to thyroid cancer, was found in the cancer group. In addition, we observed a universal expression of a recurrent read-through transcript, HALC1-COQL, in all thyroid nodules. Our results suggest that a targeted RNA-based panel provided a useful tool for fusion gene study in thyroid cancer. The role of the expected rearrangement and the unexpected read-through fusion in thyroid nodules needs to be further evaluated, as it could provide useful information for thyroid nodule differentiation.

Citation Format: Song Tian, Michelle Baird, Frank Reinecke, Jixin Deng, Mohammad Nezami Ranjbar, Raed Samara, Eric Lader. RNA fusion detection in thyroid nodules with a targeted RNA sequencing panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5408. doi:10.1158/1538-7445.AM2017-5408
Abstract 5417: The tumor suppressor klotho: A master regulator of metabolism in breast cancer
Authors: Rubinek Tami , Wolf Ido
Published: July 2017

One of the hallmarks of cancer is reprogramming of energy metabolism, characterized by a shift to aerobic glycolysis (Warburg effect). A major regulator of this phenomenon is the IGF-1/Pi3K/AKT pathway. Klotho is a transmembrane protein, which can be cleaved, shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. Klotho is a potent tumor suppressor in BC. As klotho is a potent inhibitor of the IGF-1/Pi3K/AKT pathway in BC, we hypothesized that it may revert the metabolic switch in BC cells. We first analyzed the effect of klotho on the energy sensor enzyme AMP-activated kinase (AMPK) and its down-stream effector acetyl CoA carboxylase (ACC). Overexpression of klotho, or treatment with the soluble protein, elevated AMPK and ACC phosphorylation in three BC cell lines. AMPK is activated by the tumor suppressor liver kinase B1 (LKB1) and elevated AMP/ATP ratio. Expression of a dominant negative LKB1 prevented activation of AMPK by klotho and decreased the ability of klotho to inhibit cell growth and migration, implying that klotho tumor suppressor activities are LKB1 dependent. We next analyzed the effects of klotho on critical components of glucose metabolism. Treatment with soluble klotho reduced expression of the glucose transporter GLUT1 and the key glycolytic enzymes hexokinase2 (HK2), phosphofructokinase1 (PFK1), pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase kinase 1(PDK1). Using the Seahorse analyzer we noted reduced oxidative mitochondrial metabolism and glycolysis following klotho treatment. Energy is obtained essentially in the mitochondria with the transfer of protons across the inner membrane that produces ATP. We, therefore, analyzed klotho effect on the mitochondria membrane potential and noted reduction in the mitochondria potential following treatment with klotho. Finally, we examined the effects of klotho on specific metabolites and also conducted an NMR-based metabolic profiling. In accordance with the alterations in signaling pathways and levels of glycolytic enzymes, klotho reduced glucose uptake and inhibited lactate, pyruvate and 3-hydroxy-butyrate production. Furthermore, klotho also inhibited ATP production. Taken together, our data indicate klotho as a regulator of metabolic activity in BC and suggest that reversal of the metabolic switch is a key mechanism of klotho-mediated tumor suppressor activities.

Citation Format: Tami Rubinek, Ido Wolf. The tumor suppressor klotho: A master regulator of metabolism in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5417. doi:10.1158/1538-7445.AM2017-5417
Abstract 542: Genomic and clonal characterization of immunogenic epitope landscape across The Cancer Genome Atlas
Authors: Lee HoJoon , Greer Stephanie , Grimes Susan M. , Cheong Jae-Ho , Ji Hanlee P.
Published: July 2017

Recent advances in cancer immunotherapy and genomic sequencing technologies have created promising opportunities for precision cancer medicine. Somatic mutation in coding regions of cancer genomes may lead to amino acid alterations that generate immunogenic peptides, called neoantigens. These novel peptides are tumor-specific. An individual’s own MHC genotypes restricts the presentation of these tumor-specific peptides and thus there are a limited number of candidate epitopes for immune cellular recognition. Current studies have not comprehensively studied how MHC-restricted neoantigens are present across tens of thousands of cancer. Nor have these studies determined how somatic mutations and their associated neoantigens are represented among different subclonal populations existing within individual tumors. To address these limitations, we have mapped the complex immunogenomic topology of neoantigen epitopes across 8,000 tumors representing 18 different cancer types. This neoantigen landscape accounts for how neoepitope candidates are distributed among the various clonal subpopulations existing within any given tumor.Our study utilized genomic data from the Cancer Genome Atlas (TCGA). Our automated pipeline provides results from 8,000 samples from 18 cancer types that have somatic variant calls, copy number variation, whole exome sequence, and RNA-Seq data. Identification of somatic mutations that lead to highly immunogenic antigens involved five different steps; i) in-silico translation of identified mutations, ii) expression as measured per RNA-Seq, iii) patient’s own major histocompatibility complex genotype, iv) binding affinity specific to the MHC alleles for any given patient and v) occurrence of clonal subpopulations as demonstrated by intratumoral genetic heterogeneity. We mapped the neoantigen landscape with quantitative representation of the genetic clonal diversity existing within individual tumors. In average, 2% of missense mutations from a patient are detected in RNA-Seq, with binding affinity to own HLA genotypes, and from the dominant clonal subpopulation.In summary, our study demonstrates the various relationships among the number of optimal antigens, the number of clonal subpopulations, the number of mutations, and clinical phenotypes such as tumor stage. Our automated bioinformatics process facilitates the use of exome and RNA-Seq as potential diagnostic analytic process for precision immunotherapy.

Citation Format: HoJoon Lee, Stephanie Greer, Susan M. Grimes, Jae-Ho Cheong, Hanlee P. Ji. Genomic and clonal characterization of immunogenic epitope landscape across The Cancer Genome Atlas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 542. doi:10.1158/1538-7445.AM2017-542
Abstract 5423: ZAP70 alters metabolism in chronic lymphocytic leukemic cells
Authors: Das Subhadip , Dielschneider RF , Henson Elizabeth S. , Gibson Spencer B.
Published: July 2017

Chronic lymphocytic leukemia (CLL) has the highest incidence among adults in western countries. Prognosis of CLL is highly variable but one of the biomarker zeta-chain-associated protein 70 (ZAP70) over expression is associated with aggressive disease with time to treatment being 2.6 years for ZAP70 expressing (ZAP70+) patients and 8 years for ZAP70 deficient (ZAP70-) patients. Thus ZAP70 may play a role in CLL progression. Metabolic reprograming plays a central role in cancer progression and is altered in CLL cells. However, the role of ZAP70 in metabolism in CLL cells is remains unclear. We found that ZAP70 binds to pyruvate kinase M2 (PKM2). PKM2 is key isoform of pyruvate kinase which is rate limiting enzyme of the glycolytic pathway and provides a metabolic advantage to tumour cells allowing for increased growth. Our results also indicated the PKM2 is expressed in CLL cells and binding to ZAP70 is not affected by glucose starvation or treatment with kinase inhibitors. Furthermore immunofluorescence microscopy revealed that ZAP-70 and PKM2 colocalize in CLL cells in the cytoplasm. In addition, we found ZAP70+ cells have increased rate of oxygen consumption from basal level determined by addition of inhibitor (Oligomycin A) and mitochondrial uncoupler (FCCP). Simultaneously ZAP70+ cells also have higher extracellular acidification rates. This indicates ZAP70+ CLL cells have increased mitochondrial respiratory capacity with high glycolytic rate. In addition, fluorometric analysis indicates ZAP70+ CLL cells have high glucose consumption rate towards production of lactate compared to ZAP70- CLL cells. Collectively, our data reveals a new function for ZAP70, which is to regulate the Warburg effect and may play a role in CLL progression and may identify new therapeutic targets.

Note: This abstract was not presented at the meeting.

Citation Format: Subhadip Das, RF Dielschneider, Elizabeth S. Henson, Spencer B. Gibson. ZAP70 alters metabolism in chronic lymphocytic leukemic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5423. doi:10.1158/1538-7445.AM2017-5423
Abstract 5427: Distinctions between the metabolic changes in glioblastoma cells and glioma stem-like cells following irradiation
Authors: Spehalski Elizabeth I. , Peters Cord , Tofilon Philip , Camphausen Kevin
Published: July 2017

Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in adults and, even with aggressive treatment that includes surgical resection, radiation, and temozolomide administration, prognosis is poor due to tumor recurrence. There is evidence that within GBMs a small number of glioma stem- like cells (GSCs) exist, which are thought to be radiation resistant and may be capable of repopulating a tumor after treatment. Like most cancers, GBMs largely employ aerobic glycolysis to create ATP, a phenomenon known as the Warburg effect. Experiments have shown that the cellular metabolism of GSCs differs from that of differentiated cells, making them an attractive target for novel therapeutic approaches. Much work has been done to analyze the metabolic profiles of GSCs with the goal of identifying potential therapeutic targets, but little data exists linking metabolic changes to radiation resistance. The purpose of this study is to characterize the metabolic differences between glioma stem- like cells and traditional GBM tumor cells with and without radiation treatment (IR).

To this end, we compared the metabolism of a human derived GSC line with two commonly used GBM cell lines before and after IR. At baseline, we find that glioma stem-like cells are more quiescent than GBM cells, which have higher levels of both glycolysis and oxidative phosphorylation. GBM lines show higher levels of both basal and maximal respiration, as well as basal glycolysis and glycolytic capacity than GSCs. They also express higher levels of energy, glycolysis, and TCA cycle metabolites than GSCs. Inversely, GSCs demonstrate metabolic signs of quiescence such as decreased NEAA synthesis.

After IR, the radiation- sensitive GBM tumor cell line (U251) exhibits increases in all metabolic pathways, whereas the levels of glycolytic and oxidative metabolites in the GSCs remain unchanged. All cell lines show an increase of ATP and NAD production following IR. These findings indicate that the metabolism of GSCs undergoes different alterations than that of GBM tumor cells after IR, making them an attractive target for novel therapeutic approaches in conjunction with radiation therapy. Additionally, differences in metabolic signatures between GSC lines could be useful for non-invasive diagnostic modalities such as 13C MR spectroscopy.

Citation Format: Elizabeth I. Spehalski, Cord Peters, Philip Tofilon, Kevin Camphausen. Distinctions between the metabolic changes in glioblastoma cells and glioma stem-like cells following irradiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5427. doi:10.1158/1538-7445.AM2017-5427
Abstract 544: Hypoxia response signaling is linked to TACE resistance in hepatocellular carcinoma (HCC) patients
Authors: Fako Valerie , Lee Joyce , Cheung Tan-To , Ng Irene O. , Wang Xin W.
Published: July 2017

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and outcome is dismal, due to tumor heterogeneity and lack of effective treatment options for patients with later stage disease. Transcatheter arterial chemoembolization (TACE) is the gold standard of therapy for patients with intermediate to locally advanced tumors. TACE delivers a high dose of chemotherapy directly to the tumor via the hepatic artery, followed by an embolizing agent to restrict tumor blood supply. However, tumor hypoxia is linked to alterations in metabolism, such as increased glycolysis (the Warburg effect), and can lead to enhanced cell survival. Several randomized control trials (RCTs) showed a survival benefit with TACE, but only with strict patient selection criteria. In Asia, TACE is also commonly used as adjuvant therapy after surgical resection, yet RCTs evaluating adjuvant TACE have shown conflicting results, likely due to patient selection and stratification. We hypothesize that tumor gene expression is predictive of response following TACE, and that differential cellular metabolism resembling a hypoxic phenotype prior to treatment is responsible for TACE resistance. We retrospectively analyzed gene expression data in treatment-naive tumor tissue from a cohort of Chinese patients who received TACE. Using hierarchical clustering, followed by class comparison and survival risk prediction, we identified a 14-gene signature that is predictive of response vs. non-response to TACE, as measured by overall survival, independent of other clinical variables. We found that hypoxia- and glycolysis-related genes are enriched among differentially expressed genes in TACE Responders vs. Non-Responders, and that hypoxia master regulator HIF-1α and hypoxia target gene VEGF are significantly up-regulated in Non-Responders. We determined that a key glycolysis gene is up-regulated in Non-Responders, and conversely, two rate-limiting genes involved in gluconeogenesis, the pathway opposing glycolysis, are up-regulated in Responders. We also examined metabolomic data from the TACE cohort, and found an enrichment of glycolysis-related metabolites in Non-Responders, and gluconeogenesis-related metabolites in Responders. Further investigation will be required to connect altered glucose metabolism to TACE resistance and to determine driver genes linking hypoxia and metabolism, which, together with our 14-gene signature, may serve as a stratification tool to guide personalized treatment modalities for HCC patients.

Citation Format: Valerie Fako, Joyce Lee, Tan-To Cheung, Irene O. Ng, Xin W. Wang. Hypoxia response signaling is linked to TACE resistance in hepatocellular carcinoma (HCC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 544. doi:10.1158/1538-7445.AM2017-544
Abstract 5440: miRNAs as novel therapeutic adjuvants for improving the efficacy of vincristine and radiation therapy in treating medulloblastoma
Authors: Abdelfattah Nourhan , Rajamanickam Subapriya , Subbarayalu Panneerdoss , Timilsina Santosh , Onyeagucha Benjamin , Chen Yidong , Rao Manjeet
Published: July 2017

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite recent improvements in the overall survival, only a modest percentage of patients survive Myc-driven high-risk MB. The quality of life for surviving patients is substantially reduced due to the devastating and often irreversible side effects of radiation and chemotherapy. Recently, in a large unbiased genomic screen, we uncovered a group of microRNAs (miRNAs) capable of meditating drug sensitivity in c-myc amplified high-risk MB. Our functional screen of ~1900 miRNAs identified miR-584-5p as a potent candidate that uniquely sensitizes high-risk MB to radiation as well as vincristine (VCR) (20 to 25-fold dose reduction), an anti-mitotic agent routinely administered alongside radiation and in combination with other chemotherapeutic drugs to treat medulloblastoma patients. Our studies revealed that miR-584 might act as a potent tumor suppressor as it inhibited MB growth in vivo as well as migration and invasion of c-myc amplified MB. We show that miR-584 overexpression results in defective mitosis leading to mitotic catastrophe, apoptosis, DNA damage and G2-M cell-cycle arrest in high-risk MB cells without affecting normal neural stem cell growth. Notably, we discovered that miR-584 directly regulates the expression and activity of genes including histone deacetylase 1 (HDAC1), and eukaryotic translation initiation factor 4e family member 3 (EIF4E3) that are known to play important roles in microtubule dynamics, metaphase-anaphase transition and radio-resistance. Moreover, silencing either of these two target genes resulted in significant inhibition of MB growth and enhanced sensitivity to VCR and ionizing radiation. Overexpressing miR-584 or silencing either HDAC1 or EIF4E3 also inhibited the MB stem cell proliferation and self-renewal. We report that while miR-584-5p is predominantly expressed in normal brain and cerebellum, its expression is significantly reduced in MB patient derived xenografts (PDXs). In contrast to miR-584, EIF4E3 and HDAC1 were found to be overexpressed in medulloblastoma patients. These findings are highly significant, unexpected and innovative as this miRNA and its target genes are the first to be shown to affect the therapeutic efficacy of VCR and radiation in c-myc amplified high-risk medulloblastoma.

Citation Format: Nourhan Abdelfattah, Subapriya Rajamanickam, Panneerdoss Subbarayalu, Santosh Timilsina, Benjamin Onyeagucha, Yidong Chen, Manjeet Rao. miRNAs as novel therapeutic adjuvants for improving the efficacy of vincristine and radiation therapy in treating medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5440. doi:10.1158/1538-7445.AM2017-5440
Abstract 5456: Avoiding chemotherapy resistance in squamous cell carcinomas: anticancer activities of terpenoids and their impact on the regulation of microRNAs
Authors: Hernandes Camila , Silva Bruna da , Klingbeil Maria de Fatima Guarrido , Mathor Monica Beatriz , Pereira Ana Maria Soares , Severino Patricia
Published: July 2017

MicroRNAs are small noncoding RNAs that play important roles in cellular biology. They have been implicated in pharmacogenomics by down-regulating genes that are essential for drug function. In this work we verified the potential anticancer activity of the quinone methide triterpenes maytenin and 22-β-hydroxymaytenin, as well as of a quinone methide triterpene-rich extract obtained from cultivated Maytenus ilicifolia root cells, and evaluated the associated microRNA expression following half maximal inhibitory concentration (IC50) treatment. Standard selectivity index (SI) for the isolated compounds and the root cell extract was determined by the logarithmic shift in effective concentration (IC50) between cancer cell lines and oral keratinocytes. Both isolated molecules as well as the root cell extract presented pronounced antiproliferative and pro-apoptotic activities in four cell lines derived from head and neck squamous cell carcinomas, including a metastasis-derived cell line. A positive SI, with an average 2-fold increase in potency, was detected for single agents and for the extract. MicroRNA expression profiles were assessed at 24h, 48h and 72h following treatment and an average of 100 molecules presented consistent marked variation in expression levels. Considering associations of microRNAs, genes they regulate, and the drugs effects dependent on these genes, the down-regulation of miR-193a-3p and miR-21 in treated cells is of particular interest. Both microRNAs have been involved in 5-fluorouracil and cisplatin resistance, current agents of standard chemoradiotherapy for locally advanced head and neck cancer. Squamous cell carcinoma of the head and neck is one of the most common cancer types worldwide whereas treatment options based on conventional therapies or targeted therapies under development have limited efficacy. Plant-derived products are valuable sources for the development of new therapeutic options for cancer treatment or as synergistic agents in existing regular care.

Citation Format: Camila Hernandes, Bruna da Silva, Maria de Fatima Guarrido Klingbeil, Monica Beatriz Mathor, Ana Maria Soares Pereira, Patricia Severino. Avoiding chemotherapy resistance in squamous cell carcinomas: anticancer activities of terpenoids and their impact on the regulation of microRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5456. doi:10.1158/1538-7445.AM2017-5456
Abstract 5477: Hydrogels to study ECM-oxygen gradient interactions for sarcoma cell migration
Authors: Lewis Daniel M. , Gerecht Sharon , Eisinger-Mathason T. S. Karin
Published: July 2017

Hypoxia is a critical factor in the progression and metastasis of many cancers, including soft tissue sarcomas. Frequently, oxygen (O2) gradients develop in tumors as they grow beyond their vascular supply, leading to heterogeneous areas of O2 depletion. Here, we report the impact of hypoxic O2 gradients and collagen fibers on sarcoma cell invasion and migration. We begun with O2 gradient measurements and found that large sarcoma mouse tumors (>300 mm3) contain a severely hypoxic core [≤0.1% partial pressure of O2 (pO2)] whereas smaller tumors possessed hypoxic gradients throughout the tumor mass (0.1–6% pO2). To analyze tumor invasion, we used O2-controllable hydrogels to recreate the physiopathological O2 levels in vitro. Small tumor grafts encapsulated in the hydrogels revealed increased invasion that was both faster and extended over a longer distance in the hypoxic hydrogels compared with nonhypoxic hydrogels. To model the effect of the O2 gradient accurately, we examined individual sarcoma cells embedded in the O2-controllable hydrogel. We observed that hypoxic gradients guide sarcoma cell motility and matrix remodeling through hypoxia-inducible factor-1α (HIF-1α) activation. We further found that in the hypoxic gradient, individual cells migrate more quickly, across longer distances, and in the direction of increasing O2 tension. Treatment with minoxidil, an inhibitor of hypoxia-induced sarcoma metastasis, abrogated cell migration and matrix remodeling in the hypoxic gradient. Next, we have developed hypoxic hydrogels where we can control collagen fiber density and cross-linking density independently from oxygen gradients. Using this new platform we show that an increase in fiber density in conjunction with the hypoxic oxygen gradient leads to an increase in sarcoma cell migration. This increase in migration speed was also correlated with quicker stress relaxation times of the collagen gel and larger pore size. Using these platforms we are able to create a novel 3D experimental system with control over mechanical properties (crosslinking density, binding sight density, fiber density, stress relaxation time, and stiffness) independently from oxygen concentration. Overall, we show that O2 acts as a 3D physicotactic agent during sarcoma tumor invasion and that this migration is modulated by collagen fiber density and pore size, in the surrounding matrix. We propose the O2-controllable hydrogels as a predictive system to study early stages of the metastatic process and therapeutic targets.

Citation Format: Daniel M. Lewis, Sharon Gerecht, T. S. Karin Eisinger-Mathason. Hydrogels to study ECM-oxygen gradient interactions for sarcoma cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5477. doi:10.1158/1538-7445.AM2017-5477
Abstract 5492: Comprehensive analysis of the dynamics of aberrant WNT signaling on global gene expression and nuclear structure
Authors: Brown Markus A.
Published: July 2017

The organization of the genome in the nucleus is complex and dynamic. Features of the nuclear architecture, including the spatial arrangement of genomic sequences, the structure of chromatin, and the accessibility of regulatory DNA elements modulate nuclear processes such as gene transcription DNA replication, DNA repair, RNA processing, and mRNA transport. However, the interplay between nuclear architecture and gene expression is poorly understood. We propose to elucidate the impact of silencing the prominent transcription factor, TCF7L2, the major effector of the WNT signaling pathway, on the transcriptional equilibrium and nuclear architecture of the nucleus in human colorectal cancer cells. Our multidisciplinary approach combines targeted silencing of TCF7L2, 3D-FISH, RNA-seq, and Hi-C chromosome conformation capture, in parallel with mathematical modeling to delineate structure/function relationships in the interphase nucleus. This approach will elucidate the intricacies of a dynamic cellular signaling pathway in three-dimensional space.

Citation Format: Markus A. Brown. Comprehensive analysis of the dynamics of aberrant WNT signaling on global gene expression and nuclear structure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5492. doi:10.1158/1538-7445.AM2017-5492
Abstract 5498: Autoregulation of protein kinase D1 (PRKD1) expression in prostate cancer
Authors: Nickkholgh Bita , Sivanandane Sittadjody
Published: July 2017

Protein kinase D1 (PRKD1) is down regulated in gastric, breast and prostate cancer. Several regulatory mechanisms modulate PRKD1 activity in cancers. In colon cancer, it has been shown that PRKD1 is down regulated by nuclear beta-catenin. We explored whether beta-catenin is involved in regulation of PRKD1 expression in prostate cancer as well. A CHIP assay performed using prostate cancer LNCaP cell lysate and pull down with beta -catenin antibody, which demonstrated recruitment of beta -catenin to promoter of PRKD1 gene as well as at downstream regions in the gene suggesting that beta -catenin could be involved in PRKD1 regulation. To identify the exact binding site of beta-catenin to PRKD1 gene, we carried out CHIP sequencing. The beta -catenin protein complex was bound to a 166 bp sequence near exon 2 (chr14:29899631-29899796). Because beta -catenin is a coactivator and not a known transcription factor, we performed a transcription factor promoter array, which showed that the MYC/MAX transcription factor complex may be the mediator of the regulatory effect of beta -catenin on PRKD1 expression. We validated the results by performing CHIP assay using MYC and MAX antibodies which showed recruitment of both MYC and MAX to the same beta -catenin binding site to PRKD1. In order to assess the functional impact of beta -catenin regulation of PRKD1 in prostate cancer, beta-catenin mutated at threonine

120 residue (we have previously shown that PRKD1 is the only known kinase to phosphorylate the site) demonstrated increased nuclear translocation of beta-catenin and increase down regulation of PRKD1 and increased androgen receptor (AR) activity (increased AR activity is well established to be associated with PC progression). Our data has identified a novel auto-regulatory mechanism of PRKD1 expression through beta-catenin phosphorylation.

Citation Format: Bita Nickkholgh, Sittadjody Sivanandane. Autoregulation of protein kinase D1 (PRKD1) expression in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5498. doi:10.1158/1538-7445.AM2017-5498
Abstract 5500: KMT2C directs estrogen receptor activity in normal and transformed mammary cells
Authors: Gala Kinisha , Sinha Amit , Sanchez-Vega Francisco , Chung Young Rock , Hseih James , Berger Michael , Schultz Nikolaus , Pastore Alessandro , Abdel-Wahab Omar , Chandarlapaty Sarat
Published: July 2017

Estrogen receptor alpha (ERα) is a ligand-activated nuclear receptor that regulates proliferation and differentiation in mammary epithelial cells. ERα activity is likely dependent on the actions of pioneer factors and H3K4 methyltransferases which can establish a genomic landscape permissive for ERα binding. Here, we identify the H3K4 methyltransferase KMT2C as essential for ERα activity in mammary gland development and ER+ breast cancer growth. KMT2C suppression decreases estrogen-dependent gene expression and causes H3K4me1 loss at ERα target gene enhancers. Consequently, KMT2C loss selectively suppresses estrogen-driven breast cancer proliferation. Moreover, mammary-specific Kmt2c knockout mice have defects in pubertal ductal formation similar to Esr1 deficient mice. Although KMT2C loss disrupts estrogen-driven proliferation, it conversely promotes tumor outgrowth under hormone-depleted conditions. Consistent with this, gene expression signatures of KMT2C loss are associated with poor outcomes. We conclude that KMT2C is a key regulator of ERα activity whose loss uncouples mammary phenotypes from hormone availability.

Citation Format: Kinisha Gala, Amit Sinha, Francisco Sanchez-Vega, Young Rock Chung, James Hseih, Michael Berger, Nikolaus Schultz, Alessandro Pastore, Omar Abdel-Wahab, Sarat Chandarlapaty. KMT2C directs estrogen receptor activity in normal and transformed mammary cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5500. doi:10.1158/1538-7445.AM2017-5500
Abstract 5504: Regulation of the receptor for advanced glycation end products by estrogen receptor ligands in endometrial cancer
Authors: Davies Julia , Gonzalez Deyarina , Conlan R.Steven
Published: July 2017

Endometrial cancer has numerous established risk factors including excessive unopposed estrogen exposure, tamoxifen, obesity and polycystic ovary syndrome (PCOS). These risk factors may promote inflammation, an important hallmark of cancer. Since the endometrium is a site of regular and repeated inflammation during the menstrual cycle, the regulation of inflammation may be involved in endometrial cancer. The receptor for advanced glycation end products (RAGE) stimulates numerous inflammatory signalling pathways and its expression is elevated in a number of inflammatory pathologies including diabetes and more recently cancer. This work aims to investigate the regulation of RAGE by estrogen receptor ligands in endometrial cancer. RAGE and estogen receptor isoform protein expression were examined in a patient cohort by immunohistochemistry. RAGE and ER isoform expression was assessed following estrogen receptor ligand exposure in both type I and type II endometrial cancer cell line models. ERα, ERβ, p65 and ER co-regulator recruitment to the RAGE promoter following ER ligand treatment was determined in vitro by chromatin immunoprecipitation. Data revealed (i) RAGE and ER isoform endometrial expression is altered in endometrial cancer patients and patients receiving tamoxifen compared to postmenopausal women, (ii) RAGE is expressed in type I and II endometrial cancer cell lines and its expression can be modulated by estrogen and tamoxifen exposure, and (iii) by chromatin immunoprecipitation, estrogen receptor ligands were shown to alter ERα, ERβ and ER co-regulator binding at the RAGE promoter. To conclude, results from this work suggest RAGE is associated with endometrial cancer development. Once clinically validated, RAGE and associated targets investigated in this study could be potentially used as a panel of endometrial cancer biomarkers and may also be used to indicate if patients are at risk of the adverse agonist action of tamoxifen in the endometrium.

Citation Format: Julia Davies, Deyarina Gonzalez, R.Steven Conlan. Regulation of the receptor for advanced glycation end products by estrogen receptor ligands in endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5504. doi:10.1158/1538-7445.AM2017-5504
Abstract 5541: Identification of sod and sod2 as potential prognostic biomarkers for patients with non-small cell lung cancer
Authors: Yin Chen , Wang Ren , Li Xiao-xing , Yang Yang , Zhang Mei-yin , Wang Hui-Yun , Zheng Steven X.
Published: July 2017

Backgrounds: Reactive oxygen species (ROS) is accumulated in cancer cells related to increased metabolism, which is characteristic of cancer progression. With the rapid advance of cancer research, redox regulation has drawn more and more attentions. The superoxide dismutases SOD1 and SOD2 are essential enzymes for eliminating ROS. To date, SOD1 or SOD2 have not been well characterized in non-small cell lung cancer(NSCLC). Here we compared the expression of SOD proteins between normal and cancer tissues and investigated its prognostic significance in NSCLC patients.

Method: we detected the expression of SOD1 and SOD2 in 178 NSCLC samples by immunohistochemistry and their clinical significances were analyzed. Western blotting is used to confirm the protein level of SOD1 and SOD2 in the normal and cancer tissues of NSCLC patients.

Results: The expression of SOD1 and SOD2 is significantly higher in both

adenocarcinoma or squamous cell carcinoma (p<0.0001), compared to matched adjacent normal tissues. Correlation analysis indicates that SOD1 expression is reversely associated with TNM stage, and SOD2 is negatively correlated with tumor size, tumor number and TNM stage remarkably (all p <0.05). Moreover, higher expression of SOD1 and SOD2 is correlated with better prognosis of NSCLC patients. Finally, Univariate and multivariate Cox regression modes further indicate that SOD1 and SOD2 can be used as independent prognostic factors in NSCLC patients.

Conclusion: Higher expressions of SOD1 and SOD2 in NSCLC tissues are observed compared to adjacent tissues and indicate better prognosis, suggesting that SOD proteins are potentially useful biomarkers for prognostic evaluation of patients with NSCLC.

Key Words: Reactive oxygen species; Non-small cell lung cancer; Superoxide dismutase 1/2; Prognosis.

Citation Format: Chen Yin, Ren Wang, Xiao-xing Li, Yang Yang, Mei-yin Zhang, Hui-Yun Wang, Steven X. Zheng. Identification of sod and sod2 as potential prognostic biomarkers for patients with non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5541. doi:10.1158/1538-7445.AM2017-5541
Abstract 5560: Systemic map of protein phosphatase 2A (PP2A)-regulated phosphotargets and drug responses in cancer cells
Authors: Kauko Otto , Imanishi Susumu , Kulesskiy Evgeny , Laajala Teemu D. , Yetukuri Laxmana , Padzik Artur , Jumppanen Mikael , Haapaniemi Pekka , Yadaw Bhagwan , Suni Veronika , Varila Taru , Corthals Garry , Krister Wennerberg , Aittokallio Tero , Westermarck Jukka
Published: July 2017

Despite the pivotal role of phosphatases in cancer cell signalling, systemic understanding of phosphatase targets is still at infancy. Protein phosphatase 2A (PP2A) is a human tumor suppressor complex. PP2A inhibition is a requirement for human cell transformation and PP2A regulates many cancer critical signalling pathways. Importantly, emerging data indicates that reactivation of PP2A tumor suppressor activity could provide entirely novel approach for cancer therapy. Here, we present first systemic analysis of phosphoprotein targets (dephosphorylome) regulated by PP2A in cancer cells. Based on data, PP2A regulates cancer critical signalling pathways, including entire EGFR-RAS-RAF-MEK-ERK cascade, and functions as a master regulator of MYC function. At network level, PP2A targets critical cellular processes such as chromosome organization, RNA splicing, and nuclear envelope assembly. Surprisingly, soft clustering of PP2A dephosphorylome revealed that most phospho-target residues are subject to only unidirectional regulation in cancer cells. Moreover, targets show intracellular gradient where phosphatase inhibition dominates nuclear phosphorylation balance. Since phosphoregulation is critical for cancer drug responses, dephosphorylome was correlated with cancer cell responses to over 300 drugs. Importantly, cancer therapies could be broadly classified based on their dephosphorylome, both at quantitative and qualitative manner. Finally, we demonstrate the utility of this large dataset by validating the role of PP2A in MEK inhibitor resistance in KRAS mutant cancer cells via regulation of RAF, mTor and MYC. The study presents first systemic resource to understand potential of PP2A manipulation in cancer cell signalling and drug responses. The study also uncovers generally important insights to phosphoregulation.

Citation Format: Otto Kauko, Susumu Imanishi, Evgeny Kulesskiy, Teemu D. Laajala, Laxmana Yetukuri, Artur Padzik, Mikael Jumppanen, Pekka Haapaniemi, Bhagwan Yadaw, Veronika Suni, Taru Varila, Garry Corthals, Wennerberg Krister, Tero Aittokallio, Jukka Westermarck. Systemic map of protein phosphatase 2A (PP2A)-regulated phosphotargets and drug responses in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5560. doi:10.1158/1538-7445.AM2017-5560