bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4673: DCLK1 promotes hepatocellular carcinoma via atypical β-catenin-regulated signaling and immune suppression
Authors: Ali Naushad , Chandrakesan Parthasarathy , Bronze Michael S. , Houchen Courtney W.
Published: August 2019

Introduction: Genetic lineage tracing in mouse model has revealed that mature hepatocytes are the cell of origin of injury-induced hepatocellular carcinoma (HCC), the second most common cause of cancer-related deaths worldwide. Hepatocyte plasticity, defined as the ability to change cellular identity, plays a critical role in the repair process following liver injury. Inflammatory liver diseases often lead to cirrhosis and HCC. The molecular mechanisms that regulate the decision between hepatocyte injury repair and neoplastic initiation are unclear. Doublecortin-like kinase 1 (DCLK1), a microtubule-associated kinase, is expressed during liver injury, cirrhosis, and HCC, but not in normal liver. Here, we demonstrate a novel mechanism by which DCLK1 regulates injury-associated hepatocyte plasticity and immunosuppression in HCC.

Methods: Cell culture, DEN/CCl4-induced injury in mouse livers, and humanized mouse model were used for analyzing hepatocyte plasticity and DCLK1+ cells-induced changes in hepatic cells and macrophages.

Results: DCLK1-overexpressing primary human hepatocytes formed spheroids in 3D culture. DCLK1-overexpressing hepatoma cells exhibited a 48-kDa active β-catenin with preserved hypophosphorylated N-terminus, which interacted with TCF-4, resulting in enhanced luciferase reporter activity and cyclin D1 expression. Downregulation of DCLK1 using siRNAs inhibited expression of this β-catenin fragment. DCLK1 overexpression increased phosphorylation of GSK-3β at Ser9, thus preventing its complete proteasomal degradation. Liver tissues from patients with cirrhosis and HCC expressed the 48-kDa active β-catenin, DCLK1, cyclin D1, and 80-kDa E-cadherin ectodomain, suggesting a change in epithelial polarity. DCLK1-overexpressing primary human hepatocytes transplanted into FRG mice demonstrated β-catenin activation in the transplanted hepatocytes. Injury-induced DCLK1+ epithelial cells in mouse livers expressed PD-L1. DCLK1+ human hepatoma cells induced CD206 in macrophages in a dual co-culture assay.

Conclusions: DCLK1 enhances hepatocyte plasticity, clonogenicity, and tumorigenic signaling via atypical β-catenin-dependent mechanism. DCLK1+ cells show ability to suppress the host anti-tumor immunity by expressing PD-L1 and inducing macrophage polarization.

Citation Format: Naushad Ali, Parthasarathy Chandrakesan, Michael S. Bronze, Courtney W. Houchen. DCLK1 promotes hepatocellular carcinoma via atypical β-catenin-regulated signaling and immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4673.
Abstract 4685: Cancer associated fibroblasts promote ovarian cancer chemoresistance by inducing cancer stem cells through Wnt signaling
Authors: Fang Yiming , Aziz Mohamed A. Abd El , Tiwari Kartikeya , Mitra Anirban K.
Published: August 2019

Ovarian cancer is the most lethal gynecologic malignancy and the 5th leading cause of cancer related deaths among women in the USA. Most patients eventually succumb to chemoresistant disease and the purpose of this study is to understand the mechanism of development of chemoresistance and disease relapse. Cancer stem cells (CSCs) consist of a small subpopulation in the tumor that are resistant to cytotoxic chemotherapy and cause relapse. The tumor microenvironment can potentially provide an optimal cancer stem cell niche for cancer stem cell growth and maintenance. Cancer associated fibroblasts (CAFs) are one of the main constituents of the tumor microenvironment in ovarian tumors, promoting tumor progression and chemoresistance. We have studied the potential role of CAFs in maintaining CSC population and enhancing chemoresistance with an objective to develop effective approaches to overcome chemoresistance and tumor relapse. Co-culture of high grade serous ovarian cancer cells with CAFs resulted in increased resistance to carboplatin. ALDH1A1 is a well-established marker for ovarian cancer CSCs and the ALDH+ population was significantly increased upon co-culture with CAFs. Similarly, CAFs also enhanced spheroid formation of ovarian cancer cells seeded in ultralow adhesion plates in CSC medium. Interestingly, co-culturing ALDH- ovarian cancer cells with CAFs resulted in the induction of ALDH+ cells within 6 days. Analysis of the signaling pathways activated in ovarian cancer CSCs and the gene expression profiles of ovarian cancer CAFs indicated the potential role of Wnt signaling in the productive cross-talk between CAFs and ovarian cancer CSCs. Treatment with Wnt inhibitors abrogated the induction of CSCs by CAFs. By selectively silencing porcupine, a protein involved in Wnt ligand lipidation and secretion, we further confirmed that CAF derived Wnts are responsible for the induction of CSC. Studies are ongoing to identify the specific Wnt ligand involved in the cross-talk and downstream pathways activated during CSC induction and maintenance by CAFs. Our results indicate that CAF-derived Wnt ligands are instrumental in ovarian cancer CSC growth and maintenance. In the long term, our studies will broaden the understanding of CSC maintenance by the tumor microenvironment and contribute towards the development of novel therapeutic approaches to prevent ovarian cancer chemoresistance and relapse.

Citation Format: Yiming Fang, Mohamed A. Abd El Aziz, Kartikeya Tiwari, Anirban K. Mitra. Cancer associated fibroblasts promote ovarian cancer chemoresistance by inducing cancer stem cells through Wnt signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4685.
Abstract 4687: Gastric cancer stem cell-related marker LINGO2 is associated with cancer phenotype and poor patient survival
Authors: Park Soo Been , Jo Jung Hyun , Kim Chanyang , Lee Hee Seung , Jung Dawoon E. , Song Si Young
Published: August 2019

The expression of leucine-rich repeat and immunoglobulin-like domain-containing nogo receptor-interacting protein 2 (LINGO2), has been reported in Parkinson’s disease; however, its role in other diseases is unknown. Gastric cancer is the second leading cause of cancer death. Cancer stem cells (CSC) are a subpopulation of cancer cells that contribute to the initiation and invasion of cancer. We identified LINGO2 as a CSC-associated protein in gastric cancers both in vitro and in patient-derived tissues. We studied the effect of LINGO2 on cell motility, stemness, and tumorigenicity using cells sorted based on LINGO2 expression and LINGO2-silenced cells. Tissue microarray analysis showed that LINGO2 expression was significantly elevated in advanced gastric cancers. The overall survival of patients expressing high LINGO2 was significantly shorter than that of patients with low LINGO2. Cells expressing high LINGO2 showed elevated cell motility and tumorigenicity while LINGO2 silencing reversed these properties. Silencing LINGO2 reduced AKT/MEK/ERK phosphorylation and decreased EMT-associated markers - N-cadherin and Vimentin and stemness-associated markers - OCT4 and IHH. These indicate the involvement of LINGO2 in gastric cancer initiation and progression by altering cell motility, stemness, and tumorigenicity, suggesting LINGO2 as a putative target for gastric cancer treatment.

Citation Format: Soo Been Park, Jung Hyun Jo, Chanyang Kim, Hee Seung Lee, Dawoon E. Jung, Si Young Song. Gastric cancer stem cell-related marker LINGO2 is associated with cancer phenotype and poor patient survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4687.
Abstract 4689: Subclone-specific evolution of tumor phenotypes – A framework to study subclone-specific gene expression from a combination of bulk DNA and single cell RNA sequencing data
Authors: Qiao Yi , Huang Xiaomeng , Brady Samuel , Bild Andrea , Bowtell David , Johnson William , Marth Gabor
Published: August 2019

Several approaches are now available for subclonal reconstruction of heterogeneous tumor biopsies from somatic variant allele frequencies measured in bulk DNA sequencing datasets, including our own SubcloneSeeker program. However, to understand how chemoresistance, relapse, or metastasis evolves at a subclonal level, we also need to study the molecular phenotype corresponding to each subclone. Single-cell RNAseq technologies now allow one to study the transcriptomic characteristics and phenotypic behaviors of individual cells, but new algorithms are needed to link these cells to the genomically defined tumor subclones they represent.

Here we present a computational approach to make such cell assignments, using a combination of bulk DNA sequencing data (WGS or WES), and single cell RNA sequencing (scRNAseq) collected from the same tissues. Subclones constructed from bulk DNA sequencing data are defined by specific combinations of somatic mutations (SNVs and CNVs). Our algorithm uses the scRNA-seq reads to assess which of these subclone-defining mutations are present in each individual cell, then utilizes a Bayesian probabilistic framework for making the cell-to-subclone assignment. This framework overcomes the challenges of sparse scRNA-seq read coverage, and maximizes the accuracy and efficiency of cell assignment.

We have successfully applied this method to multiple longitudinal and metastatic cancer patient datasets, representing both WES and WGS bulk DNA sequencing, as well as datasets collected using the 10X Chromium and Fluidigm C1 technologies. Our algorithm uses both somatically acquired CNV and SNV events in the tumor for cell assignment. Using our approach, as many of 80% of tumor cells could be assigned to specific subclones, enabling comparative gene expression and pathway analysis across subclones.

Citation Format: Yi Qiao, Xiaomeng Huang, Samuel Brady, Andrea Bild, David Bowtell, William Johnson, Gabor Marth. Subclone-specific evolution of tumor phenotypes – A framework to study subclone-specific gene expression from a combination of bulk DNA and single cell RNA sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4689.
Abstract 4698: Development of a CRISPR-Cas9D10A targetable, high-complexity, single-cell barcoding approach for capture of treatment resistant subclones from heterogeneous cancers
Authors: Zhang Ze-yan , Ezhilarasan Ravesanker , Ding Yingwen , Wang Qianghu , Yang Jie , Long Lihong , Verhaak Roel G. , Sulman Erik P.
Published: August 2019

Cancers are composed of heterogeneous cell populations and the clonal evolution of these cells is one of the key reasons for treatment resistance and tumor recurrence. A fundamental challenge in studying clonal evolution in these tumors is the difficulty in capturing the phenotype-associated (e.g. treatment resistant) sub-populations from the heterogeneous population. We developed an approach to individually barcode and isolate specific cell subpopulations by constructing a Cas9D10A and paired-gRNA targetable unique reporter (CAPTURE) barcoding library with up to 36 million unique barcodes. This approach enabled us to uniquely barcode > 1 million cells, track barcode distribution following treatment and then isolate the resistant subpopulation using the subpopulation-specific barcode. Proof of principle studies showed that specific barcoding cells, even at as low as 0.1%, could be efficiently isolated using barcode targeting and cell sorting. Applied our barcoding approach, we found that A375 cell acquired BRAF inhibitor resistance both from pre-exiting and late-emerging mechanisms. Colony formation experiments showed that the isolated subpopulations were indeed resistant clones. Whole exome, transcriptome and methylome analysis were applied to study the captured subpopulations. Our CAPTURE barcoding approach will enable the identification of the both pre-exiting and late-emerging genetic or epigenetic changes driving treatment resistance.

Citation Format: Ze-yan Zhang, Ravesanker Ezhilarasan, Yingwen Ding, Qianghu Wang, Jie Yang, Lihong Long, Roel G. Verhaak, Erik P. Sulman. Development of a CRISPR-Cas9D10A targetable, high-complexity, single-cell barcoding approach for capture of treatment resistant subclones from heterogeneous cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4698.
Abstract 4703: Single Cell CNV End to End workflow to uncover tumor heterogeneity in a murine breast cancer model
Authors: Gibbs Lee D. , Qian Jing , Webb Michelle , Chang Sharon , Manojlovic Zarko , Velazquez Enrique I. , McEachron Troy , Craig David W. , Carpten John D.
Published: August 2019

Background: Tumor heterogeneity is a hallmark of cancer and can have significant impact on identifying drivers, including those that may be therapeutically relevant. Although, the traditional sequencing of bulk tumor specimens provides invaluable information, sequencing of individual cells provides a true representation of the cellular heterogeneity that exists within a cancer. Novel technologies such as single cell copy number variation (scCNV) enable copy number profiling at a single cell resolution. Single Cell CNV generates genome wide sequencing libraries from single cells to reveal tumor heterogeneity and evolution of subclonal populations.

Methods: In this study, we utilized a scCNV assay to assess heterogeneity within the 4T1 murine breast tumor model. To accomplish this we have developed an end-to-end workflow including tissue dissociation, single cell library preparation, sequencing and data analysis. We injected 4T1 cells into the mammary fat pad of 4-6 week old BALB/c mice. Mice were sacrificed at 21 days and tumors were removed from the injection site and dissociated using Milteny’s Biotec Octo Dissociator. We utilized the 10X Genomics Chromium system to partition single cells and prepare sequencing libraries in parallel such that all DNA fragments produced within a partition are barcoded. Following library construction, the Illumina Novaseq 6000 system was used to sequence single cell libraries. Additionally, single cell libraries from a fraction of the 4T1 cells that were injected into the mammary fat and non-metastatic isogeneic cell line variants, 67NR and 168FARN, were also sequenced.

Results: We observed a pair of amplifications (>8 copies) on chromosome 9 and three segments of amplification on chromosome 15 that were conserved throughout the injection site tumor cells. Interestingly, the amplifications on chromosome 15 encompass one of the most commonly observed breast cancer oncogenes, Myc, and the cell migration regulator, Skp2. Further, we observed three unique cluster of tumors cells which suggests the possibility of detecting major and minor clonal populations using this technology.

Conclusion: Our findings suggest scCNV will help us to improve characterizations of the entirety of a patient’s disease to uncover potential clones that are primed to induce metastasis, drug resistance, and relapse.

Citation Format: Lee D. Gibbs, Jing Qian, Michelle Webb, Sharon Chang, Zarko Manojlovic, Enrique I. Velazquez, Troy McEachron, David W. Craig, John D. Carpten. Single Cell CNV End to End workflow to uncover tumor heterogeneity in a murine breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4703.
Abstract 4715: Combination of Tacedinaline and EHMT2 inhibition increases breast cancer cell death involving BIRC5 repression and GADD45A induction
Authors: Chu Pei-Yi , Lai Ji-Ching , Hou Ming-Feng , Lin Chang-Shen
Published: August 2019

The genomes of cancer cells are different from those of normal cells. These differences include genetic and epigenetic alterations, such as variations of DNA methylation and histone modifications, which cause differential gene expression, increased cell growth/migration capacity, and may induce resistance to anticancer therapy. Thus, epigenetic modulation may have a chance to correct abovementioned cancer cell’s aberrations in gene expression and cell behaviors, resulting in eradication of primary and/or suppression of refractory cancers.

Tacedinaline (CI-994) and UNC0638 are specific inhibitors of class I histone deacetylase (HDAC) and histone methyltransferase EHMT2 (G9a), which cause an increase of histone acetylation and a decrease of histone H3 dimethylation at lysine 9, respectively. Here we examined the therapeutic effect of CI-994 and UNC0638 combination on the triple-negative (MDA-MB-231) and estrogen receptor-positive (MCF-7) breast cancer cell lines.

The results showed that the combination of CI-994 and UNC0638 was able to induce more severe cell death than either agent alone. Although the sub-G1 fraction and Annexin V-positive cells were increased, CI-994 and UNC0638 did not significantly induce the activities of caspases 3, 8 and 9, suggesting the involvement of non-canonical and caspase-independent cell death mechanism. Instead, CI-994 suppressed the expression of BIRC5 (Survivin, a member of the inhibitor of apoptosis protein) and, in combination with UNC0638, induced the expression of GADD45A (involved in stress-induced apoptosis).

Mechanistically, combination of CI-994 and UNC0638 resulted in the changes of epigenetic marks, such as acetylation and methylation of histone H3, at the loci of BIRC5 and GADD45A, which were correlated with their altered gene expressions. Genetic knockdown of BIRC5 expression in MCF-7 decreased cell viability, supporting the pro-survival role of BIRC5 in breast cancer cells.

Importantly, the targets of CI-994 (HDAC1, HDAC2) and UNC0638 (EHMT2), as well as BIRC5 were overexpressed in breast cancer specimens, especially those of triple-negative breast cancer (TNBC), based on the analyses of The Cancer Genome Atlas dataset. Meanwhile, the expression of GADD45A was downregulated. BIRC5 upregulation and GADD45A downregulation in breast cancer were associated with patient’s overall survival.

These results suggest that Tacedinaline and UNC0638, or targeting the class I HDAC and EHMT2, may be a potential strategy to treat breast cancer including TNBC.

Citation Format: Pei-Yi Chu, Ji-Ching Lai, Ming-Feng Hou, Chang-Shen Lin. Combination of Tacedinaline and EHMT2 inhibition increases breast cancer cell death involving BIRC5 repression and GADD45A induction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4715.
Abstract 4734: Response of bladder and colon cancer cells to combined treatment with bromodomain and histone deacetylase inhibitors
Authors: Lea Michael A. , Patel Niyati , desBordes Charles
Published: August 2019

Since bromodomains of proteins bind acetylated histone lysine side chains while histone deacetylase inhibitors increase the level of histone acetylation, it seemed possible that bromodomain inhibitors might counteract some cellular responses to the action of histone deacetylase inhibitors. We have examined this possibility in bladder and colon cancer cells treated with combinations of bromodomain and histone deacetyalase inhibitors. Actions on growth and on the induction of alkaline phosphatase activity by histone deacetylase inhibitors were investigated. Compounds studied were the bromodomain inhibitors JQ1, I-BET763, olinone and RVX208 and the histone deacetylase inhibitors butyrate, RGFP966 and valproate. The cells studied were ones in which alkaline phosphatase activity is induced by histone deacetylase inhibitors. They were bladder cancer lines 5637, HT1197 and HT1376 and colon cancer lines Caco-2 and HT29. The combination that most clearly showed decreased induction of alkaline phosphatase activity by a bromodomain inhibitor was butyrate plus JQ1. If this action represents blocking combination of bromodomain-containing transcription factors with acetylated histones one might anticipate that inhibitory effects of histone deacetylase inhibitors on growth might be mitigated by combination with bromodomain inhibitors. However, the tendency was for additive effects on growth inhibition in accord with some data in the literature. Of the bromodomain inhibitors that were studied, JQ1 was the most effective as a growth inhibitor and olinone was the least effective. The mechanism for additive effects of bromodomain and histone deacetylase inhibitors on growth remains to be defined but antagonistic effects on cell differentiation appear possible.

Citation Format: Michael A. Lea, Niyati Patel, Charles desBordes. Response of bladder and colon cancer cells to combined treatment with bromodomain and histone deacetylase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4734.
Abstract 4752: Exon-16-skipping HER2 contributes to osimertinib-resistance through Src-independent manner in EGFRL858R/T790M-positive non-small-cell lung cancer
Authors: Hsu Chia-Chi , Liao Bin-Chi , Liao Wei-Yu , Markovets Aleksandra , Stetson Daniel , Thress Kenneth , Yang Chih-Hsin
Published: August 2019

Osimertinib is current recommended treatment for EGFR T790M-positive NSCLC following progression on prior EGFR tyrosine kinase inhibitor therapy. However, acquired resistance to osimertinib therapy inevitably developed after a period of effective treatment. We had a patient of EGFR L858R/T790M-positive NSCLC who initially responded to osimertinib therapy but eventually developed resistance. Plasma cell-free DNA analysis disclosed exon-16-skipping HER2 (HER2Δ16) in one osimertinib treated patient. HER2Δ16 was regarded as an oncogenic driver in breast cancer and has never been reported in lung cancer before. Therefore, it’s intriguing to investigate the role of HER2Δ16 in osimertinib resistance in NSCLC. The purpose of this study is to explore the mechanism of HER2Δ16-mediated resistance to osimertinib and to explore strategies to overcome the resistance.

We constructed and established H1975 cells stably expressing either vehicle, WT-HER2 or HER2Δ16. We observed that without osimertinib treatment, there was no difference in cell growth and the response to EGF-stimulation. However, when treated with osimertinib, H1975-HER2Δ16 cells were more resistant under normal and EGF-stimulating conditions compared to vehicle or WT-HER2 expressing cells. Moreover, osimertinib treatment in H1975-HER2Δ16 cells had less inhibitory effect on phosphor-ERK and phosphor-AKT compared to vehicle or H1975-WT-HER2 cells. Interestingly, co-treatment of osimertinib with Src-kinase inhibitor, dasatinib, failed to reverse the resistance, indicating that HER2Δ16-driven

osimertinib-resistance was independent of Src-kinase. Finally, we revealed that combination of osimertinib with the HER2 small molecular inhibitor, afatinib, can suppress cell growth in H1975-WT-HER2 and H1975-HER2Δ16 cells.

In summary, we have shown that HER2Δ16, may be a resistance mechanism to osimertinib in EGFR mutated NSCLC and that HER2Δ16-driven resistance was Src-kinase independent. Moreover, we found the HER2Δ16-driven resistance could be rescued by combination of osimertinib with afatinib.

Citation Format: Chia-Chi Hsu, Bin-Chi Liao, Wei-Yu Liao, Aleksandra Markovets, Daniel Stetson, Kenneth Thress, Chih-Hsin Yang. Exon-16-skipping HER2 contributes to osimertinib-resistance through Src-independent manner in EGFRL858R/T790M-positive non-small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4752.
Abstract 4795: Oxidative stress by proguanil suppresses breast tumor growth
Authors: Gupta Nehal , Srivastava Sanjay
Published: August 2019

Breast cancer is one of the most common malignancies and the second leading cause of cancer related mortality among women in U.S., thus developing new strategies to control breast cancer is an important mission. Repurposing of old drugs as new anti-cancer drugs is important as it can save time and cost of drug development. Proguanil is used in combination with atovaquone for the treatment of malaria. In this study, we determined the anticancer effects of proguanil in breast cancer cells. Proguanil significantly reduced the viability of MDA-MB-231, HCC1806 and MCF-7 breast cancer cells with IC50 of 42μM, 44μM and 40μM respectively after 72 h of treatment and induced apoptosis as exhibited by FITC/Annexin assay and cleavage of caspase 3 as well as PARP. Proguanil also reduced the survival of several patient-derived cells with IC50 in the range of 30-40μM. The anti-cancer effects of proguanil were associated with the generation of ROS and persistent disruption of mitochondrial membrane potential. ROS generation by proguanil was about 2-3 fold more as compared to control in MDA-MB-231 and HCC1806 cells. The generation of ROS was inhibited when the cells were pretreated with a general antioxidant N-acetyl cysteine (NAC). Moreover, exposure of MDA-MB-231 and HCC1806 cells to proguanil was also associated with increased expression of Bax, p-H2AX, c-caspase9 and down-regulation of bcl-2 and survivin. We further evaluated the oxygen consumption rate with proguanil treatment in breast cancer cells using flux analyzer. Our results showed significant decrease in the mitochondrial respiration rate and ATP production rate after proguanil treatment. Furthermore, 20mg/kg proguanil when given orally suppressed the growth of 4T1 breast tumors in female Balb/c mice by 55%. Tumors from proguanil treated mice demonstrated increased apoptosis, which was related to the increased expression of p-H2AX and Bax. Taken together, these results show that proguanil is an effective inhibitor of in vitro and in-vivo growth of breast cancer cells. These findings provide the rationale for further clinical investigation of proguanil against breast cancer.

Citation Format: Nehal Gupta, Sanjay Srivastava. Oxidative stress by proguanil suppresses breast tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4795.
Abstract 4797: A novel Diiminoquinone targets colorectal cancer stem cells
Authors: Monzer Alissar , Jabotian Nayri , Zhu Jie S. , Kurth Mark J. , Kheir Wassim Abou , Haddadin Makhlouf , Gali-Muhtasib Hala
Published: August 2019

5-Fluorouracil (5-Fu) remains the standard chemotherapy for metastatic colorectal cancer, but drug resistance and unpredictable cardiotoxicity limit its effectiveness. The high recurrence rates and common resistance are thought to be due to a population of self-renewing cancer stem cells (CSCs). In this study, we synthesized four novel heterocyclic compounds that are similar in structure with quinones and tested their anticancer activity against HCT116 human colon cancer cells in 2D monolayer and 3D sphere cultures. In 2D, all compounds caused significant inhibition of colon cancer cell viability at concentrations non-cytotoxic to normal human FHs74Int intestinal cell lines. In 3D cultures, these heterocycles eradicated the self-renewal ability of the highly resistant cancer stem cells and inhibited colon sphere formation in first generation (G1), as well as subsequent generations. This study represents the first documentation of the activity of these novel heterocyclic compounds, particularly compound 6a, abbreviated as DIQ-3, which we propose to be an effective treatment strategy to prevent colon cancer recurrence by targeting colorectal CSCs. Our findings provide the basis for suggesting these non-toxic and stable compounds for additional testing against cancer.

Citation Format: Alissar Monzer, Nayri Jabotian, Jie S. Zhu, Mark J. Kurth, Wassim Abou Kheir, Makhlouf Haddadin, Hala Gali-Muhtasib. A novel Diiminoquinone targets colorectal cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4797.
Abstract 4798: The Ashitaba (Angelica keiskei) chalcones 4-hydroxyderricin and xanthoangelol suppress melanomagenesis by targeting BRAF and PI3-K
Authors: Zhang Tianshun , Wang Qiushi , Ashida Hitoshi , Bode Ann , Dong Zigang
Published: August 2019

Malignant melanoma is an aggressive tumor of the skin and still lacks effective preventive and therapeutic approaches. In melanoma, both the BRAF/MEK/ERK and PI3-K/AKT signaling pathways are constitutively activated though multiple mechanisms, which result in cell cycle progression and prevention of apoptosis. Therefore, the development of novel strategies for targeting BRAF and PI3-K are of utmost importance. In the current study, we found that the Ashitaba (Angelica keiskei) chalcones, 4-hydroxyderricin (4HD) and xanthoangelol (XAG), suppressed melanoma carcinogenesis by directly targeting both BRAFV600E and PI3-K, which blocked the activation of downstream signaling. This led to the induction of G1 phase cell cycle arrest and apoptosis in melanoma cells. Importantly, 4HD and XAG dramatically attenuated tumor incidence and volume in the BRAF-activated Pten-deficient melanoma mouse model. Our findings suggest that 4HD and XAG are promising chemopreventive or potential therapeutic agents against melanomagenesis that act by targeting both BRAF and PI3-K, providing hope for rapid clinical translation.

Citation Format: Tianshun Zhang, Qiushi Wang, Hitoshi Ashida, Ann Bode, Zigang Dong. The Ashitaba (Angelica keiskei) chalcones 4-hydroxyderricin and xanthoangelol suppress melanomagenesis by targeting BRAF and PI3-K [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4798.
Abstract 4802: In vitro characterization of TP-2846: a novel tetracycline antileukemia agent
Authors: Fyfe Corey , Newman Joseph , Bhargava Ajay , Ballesteros Juan , Robles Alicia , Sun Cuixiang , Xiao Xiao-Yi , Dumas Jacques
Published: August 2019

Recently published studies suggest two hypotheses for the mechanism of action (MOA) of tetracyclines’ anticancer activities: a) disruption of mitochondrial translation (Škrtić, Cancer Cell (2011) 20(5): 10.015); b) regulation of the miRNA-199b-5p-HES1-AKT pathway (Yang, Mol Cancer Ther (2016) 15(3): 1535-7163.MCT-15-0709). Upon treatment with TP-2846, suppressed levels of cytochrome oxidase-1 (COX-1) levels were demonstrated in Western blots. The COX-1 mRNA accumulated approximately 1,000-fold over background levels after 48 h incubation. Furthermore, TP-2846 displayed sub-µM inhibitory activities to mitochondrial translation (IC50 = 0.71 µM) and ribosomal translation both in E. coli and in eukaryotes (IC50 = 0.56 µM and 3.75 µM, respectively).

In addition, TP-2846 led to accumulation of the micro RNA miR-199b in MV4-11 and HL-60 cells after 24 h incubation. TP-2846 had minimal IC50 shift (Resistance Factor (RF) = 1.53) against K-562/ADR, a leukemia cell line overexpressing P-glycoprotein (Pgp), while daunorubicin displayed an RF value of 3.67 in the same assay. TP-2846 was also studied ex vivo using bone marrow samples from 15 AML patients, including those resistant to AraC, and displayed potent activity in all samples (EC50 = 150-250 nM), while anthracyclines had similar activity (EC50 = 100-600 nM) and AraC was 10x less potent (EC50 = 2.3 µM) in the same ex vivo assay.

In summary, TP-2846 exhibits both anticancer MOAs described in the literature for tetracyclines, retains antiproliferation activity in resistant leukemia cell lines, and demonstrates promising ex vivo activity in bone marrow samples from AML patients. This data indicates that further investigations in vitro and in vivo are warranted (see accompanying abstract “In vivo activities of TP-2846: a novel tetracycline antileukemia agent”).

Citation Format: Corey Fyfe, Joseph Newman, Ajay Bhargava, Juan Ballesteros, Alicia Robles, Cuixiang Sun, Xiao-Yi Xiao, Jacques Dumas. In vitro characterization of TP-2846: a novel tetracycline antileukemia agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4802.
Abstract 4806: Development of a novel antitumor agent NCT compound for the treatment of non-small cell lung cancer
Authors: Hyun Seung Yeob , Le Huong Thuy , Yong Young-Sik , Min Hye-Young , Lee Jeewoo , Lee Ho-Young
Published: August 2019

Despite the development of advanced therapeutic regimens such as molecular targeted therapy and immunotherapy, the 5-year survival of patients with lung cancer is still less than 20%, suggesting the need to develop additional treatment strategies. Here we show the efficacy and biological mechanism of a novel antitumor agent, NCT compound. NCT compound exhibited significant inhibitory effects on the viability and colony formation of non-small cell lung cancer (NSCLC) cells and those carrying resistance to chemotherapy by inducing apoptosis. NCT compound markedly suppressed the migration of NSCLC cells. Consistently, NCT compound significantly suppressed tumor growth in a xenograft model. NCT compound showed minimal effects on the viability of normal cells and no overt toxic effects in mice, suggesting minimal toxicity of NCT compound. Further mechanistic studies revealed that NCT compound downregulated the activation of signaling pathway associated with cell proliferation and survival and the expression of epithelial-mesenchymal transition (EMT)-related markers. These results suggest the potential of NCT compound as an antitumor agent.

Citation Format: Seung Yeob Hyun, Huong Thuy Le, Young-Sik Yong, Hye-Young Min, Jeewoo Lee, Ho-Young Lee. Development of a novel antitumor agent NCT compound for the treatment of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4806.
Abstract 4810: Olive oil-derived (-)-oleocanthal for prevention of breast cancer recurrence
Authors: Siddique Abu Bakar , Ayoub Nehad M. , Sayed Khalid A. El
Published: August 2019

Despite progress in BC therapy and improved survival rates, several breast cancer (BC) patients have poor recurrence-free rates. More than three million BC survivors are currently at the risk of disease recurrence without feasible preventive options. Surgical excision of early-stage confined breast tumors commonly used to minimize subsequent metastasis. Neoadjuvant and adjuvant therapeutic agents usually cannot prevent BC recurrence. There is a dire need to discover novel recurrence and metastasis inhibitory entities because clinical trials on early-stage cancer patients survival and metastases/recurrence reduction endpoint is not financially feasible and need large patients number. Mediterranean populations have less colon and breast cancers incidence due to their dietary consumption of significant amounts of phenolics-rich extra-virgin olive oil (EVOO). (-)-Oleocanthal (OC) is the most bioactive EVOO phenolic with diverse activities and exceptional in vivo potency. Validated molecular targets of OC in BC include the inhibition of the activation of the RTKs c-Met and HER2 and modulation of estrogen receptors (ER). Met/HER2/ER amplification is implicated in activation of quiescent breast tumor cells, repopulation, subsequent recurrence and relapse. This study reports the potent ability of four-weeks oral 10 mg/kg OC treatments to inhibit more than 90% of local and regional triple negative and hormone-dependent breast tumors recurrence after the surgical excision of orthotopically xenografted primary breast tumors. Oral OC treatments also potently inhibited recurrence after surgical excision of breast tumors subjected to neoadjuvant therapies with either lapatinib (50 mg/kg, 5X/week, 4 weeks) or paclitaxel (4.5 mg/kg, ip, 3X/week for 6 weeks) in athymic orthotopic nude mice models. This was associated with significant reductions of p-c-Met and p-HER2 levels in treated animal tumors by Western blotting. OC-treated mice sera completely lacked the BC recurrence marker CA 15-3, unlike vehicle treated controls which showed significantly high CA 15-3 levels. OC treatment did not show any observable toxicity and did not affect animals body weight over the experiment course. OC is a novel breast cancer recurrence inhibitory dietary supplement lead with excellent clinical applications potential.

Note: This abstract was not presented at the meeting.

Citation Format: Abu Bakar Siddique, Nehad M. Ayoub, Khalid A. El Sayed. Olive oil-derived (-)-oleocanthal for prevention of breast cancer recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4810.
Abstract 4840: Characterization of URST1 as a biomarker and therapeutic target for precision medicine of lung cancer
Authors: Takano Atsushi , Miyagi Yohei , Daigo Yataro
Published: August 2019

This study aims to identify novel biomarkers and therapeutic targets for lung cancer. We initially screened genes that overexpressed in the majority of lung cancers by using our original gene expression profile database. During this process, we identified up-regulated in solid tumor 1 (URST1) as a candidate. Immunohistochemical staining showed that URST1 was expressed in 231 (64.5%) of 358 non-small cell lung cancer (NSCLC) that had undergone curative surgery. Strong URST1 expression was associated with poor prognosis for NSCLC patients (P = 0.0003 by log-rank test). In addition, multivariate analysis showed that strong URST1 expression was an independent prognostic factor for NSCLC patients. Reduction of URST1 expression by siRNA or treatment with selective URST1 inhibitor significantly suppressed cancer cell growth partly through cell cycle arrest at G2/M phase and mitotic cell death as detected by flow cytometric analysis and live cell imaging. Our findings suggest that URST1 could be a potential biomarker and therapeutic target for developing precision medicine of lung cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Atsushi Takano, Yohei Miyagi, Yataro Daigo. Characterization of URST1 as a biomarker and therapeutic target for precision medicine of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4840.
Abstract 4856: Suppression of PRKCI gene-amplified ovarian cancer using aptamer-delivered siRNA
Authors: Rehmani Hina
Published: August 2019

Ranked fifth in cancer death among women, ovarian cancer cure rates have remained at 30% over the past two decades. Treatment options have steadily followed the same methodology, surgery and then first-line chemotherapy. With a 70% relapse rate within the first two years of diagnosis and limited secondary options, acquiring more targeted treatments is imperative. Copy number aberrations, more than mutations, are commonly found in high grade serous ovarian cancer. Copy number gains of the Protein Kinase C iota (PRKCI) gene are found in over 33% of ovarian cancer patients. According to the TCGA database, patients with PRKCI gene amplification have a significantly poor overall survival than those that do not. With this information and the fact that it is a previously identified oncogene in ovarian cancer, Protein Kinase C iota (PKCi) serves as an excellent target. Unfortunately, the Protein Kinase C iota isoform shares an overall homology of 72% with the Protein Kinase C zeta isoform which makes selective targeting of the iota form a challenge. Previous publications have used gold-based compounds to block atypical PKC family members from interactions that are crucial for apical-basal polarity. We found, however, that these compounds exhibit no specific selectivity for PRKCI gene amplified ovarian cancer cell lines.To specifically target PKCi, we have created an RNA-based aptamer that contains a siRNA sequence against the PRKCI mRNA and EpCAM moieties on either end. The aptamer efficiently becomes internalized after binding to the cell surface EpCAM via clathrin-dependent endocytosis. Once inside, the aptamer is processed by Dicer to release the two unique siRNA duplex intermediates. The guide strand of the siRNA loads into the RNA-induced silencing complex (RISC) complex and Argonaute (Ago) protein family members mediate PRKCI mRNA silencing.We hypothesize that PRKCI gene amplification status offers a unique opportunity to stratify patients into different risk categories and RNA-aptamer targeting of PRKCI can provide therapeutic benefits, by impairing ovarian cancer cell proliferation and tumorigenesis. We expect this approach to provide potent and selective anti-tumor and anti-metastasis activity compared to targeting using other pharmaceutical options.

Citation Format: Hina Rehmani. Suppression of PRKCI gene-amplified ovarian cancer using aptamer-delivered siRNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4856.
Abstract 488: Direct real-time visualization and quantification of T cell receptor dynamics
Authors: Rosenberg Jillian N. , Cao Guoshuai , Borja-Prieto Fernanda , He Yanran , Schreiber Hans , Huang Jun
Published: August 2019

The mechanism by which a T cell utilizes its T-cell receptors (TCRs) to recognize foreign peptide major histocompatibility complexes (pMHCs) has not yet been fully deciphered. Much contention still exists surrounding TCR nanostructures before, during, and after synapse formation. Accurate measurements of single TCR location, diffusion/trafficking, clustering, and signaling at the live T-cell membrane have been hindered by the microscopy techniques available. Recently developed Lattice Light-Sheet Microscopy uses a structured light sheet to excite fluorescence in successive planes of a living T cell such that it can record 4D (x, y, z directions and time) images with exceptionally high temporal resolution (10ms/frame, ~1s/volume), finally allowing for precise single molecule tracking. Here we show that TCRs pre-exist in small microclusters prior to synapse formation in both naïve and stimulated resting CD4+ T cells. These clusters are very fast-moving, explaining the previous difficulties of tracking TCR motion and revealing a fast responding mechanism for antigen recognition. Upon encountering antigens, these smaller TCR nanoclusters traffic globally and quickly to the cell interface, aggregate into larger microclusters, and form the immunological synapse. Finally, we compare and contrast the receptor dynamics of CAR-T cells with those of naturally occurring T cell receptors. These findings precisely quantify TCR and CAR dynamics three-dimensionally, suggesting new mechanistic details of the speed and accuracy of TCR signaling and providing new information upon which to base future immunotherapies.

Citation Format: Jillian N. Rosenberg, Guoshuai Cao, Fernanda Borja-Prieto, Yanran He, Hans Schreiber, Jun Huang. Direct real-time visualization and quantification of T cell receptor dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 488.
Abstract 4888: Nuclear localization of antizyme inhibitor may be a marker for aggressiveness of prostate cancer
Authors: Ghalali Aram , Rice James M. , Wang Liangzhe , Wu Chin Lee , Rogers Michael S. , Zetter Bruce
Published: August 2019

Antizyme suppresses cell cycle by inhibiting the polyamine synthesis through binding to the rate limiting enzyme ornithine decarboxylases (ODC). High levels of ODC have been reported in several forms of cancer, among them prostate cancer. Antizyme Inhibitor (AZIN) binds to antizyme and thereby blocks its inhibitory effect on ODC. Here, we have measured the expression and localization of AZIN in 202 prostate cancer specimens, along with 26 adjacent benign samples and found that nuclear localization of AZIN is associated with significantly lower survival. Upregulation and nuclear localization of AZIN have been observed in several cancers, as has editing of the AZIN1 mRNA. Others have hypothesized that the RNA- edited AZIN (edAZIN) may have an increased affinity to antizyme and that could explain the association of edAZIN to the various cancers. We have studied the mechanism behind the nuclear localization of AZIN and found the single base pair substitution caused by RNA editing is sufficient to result in nuclear localization of the protein in all cell types tested. To determine if the nuclear localization might result from increased antizyme-edAZIN affinity, we developed fluorescent protein FRET sensor for protein-protein interaction using Clover-AZIN and antizyme-mRuby2 fusion proteins. Unexpectedly, we found that the editing event modestly decreases edAZIN affinity for antizyme, notwithstanding increased interaction in vivo. Thus, the data indicate that the change in protein localization to the nucleus may be more important to oncogenic function than the actual degree of binding to antizyme. Other functional differences between edAZIN and AZIN might be explained by altered kinetics of binding, by the contribution of an additional adapter protein which modulates the intracellular antizyme:AZIN complex, or by competition for AZIN binding by other partners whose interaction is affected by the editing event. Finally, we identified AZIN and edAZIN interacting proteins by using tandem affinity purification and LC-MS-MS analysis. Among interacting proteins, we identified a complex containing two isoforms of nuclear actins (ACTG1 and ACTA2) and Myosin-9 that may be the driving force behind the nuclear shuttling of edAZIN.

Citation Format: Aram Ghalali, James M. Rice, Liangzhe Wang, Chin Lee Wu, Michael S. Rogers, Bruce Zetter. Nuclear localization of antizyme inhibitor may be a marker for aggressiveness of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4888.