bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A62: Canine pedigree analysis as a model of hereditary breast cancer
Authors: Lloveras-Fuentes Carlos , Goebel Katie , Merner Nancy D.
Published: August 2018

Genetic homology with humans and breeding practices that limit intrabreed heterogeneity make canines a useful genetic model for identifying heritable disease markers that have parallels in humans. Ultimately, the homogeneity of individual canine breeds is advantageous in identifying the modes of inheritance for human disorders that have resisted investigative efforts for decades. Particularly few genetic research studies have focused on identifying inherited risk factors for canine mammary tumor (CMT), and as such there has been little exploration of the potential overlaps between the ~65% of human hereditary breast cancer cases that are currently genetically unsolved.

DNA samples from 85 CMT-affected canines were extracted from blood/buccal swabs for whole-genome sequencing. These included 32 different canine breeds from six of the seven American Kennel Club (AKC) recognized breed groups; the most represented breeds were Golden Retrievers (n=20), Siberian Huskies (n=8), and Standard Schnauzers (n=7). The cohort also included 3 male CMT cases, which is a hallmark of hereditary CMT and comparable to hereditary breast cancer incidence in human males. Furthermore, pedigree analyses identified dogs within the CMT cohort who stem from a breed-specific common ancestor, and this indicates increased genetic homogeneity within a breed. Currently, these dogs exist in twelve different canine pedigrees corresponding to twelve different breeds and representing 72% of the CMT cohort. The most impressively linked pedigrees contain 20/20 Golden Retrievers, 7/8 Siberian Huskies, 7/7 Standard Schnauzers, 5/5 Welsh Springer Spaniels, 4/4 Newfoundlands, 4/4 Golden Setters, 3/3 Dalmatians, and 3/3 Doberman Pinschers. These CMT pedigrees highlight the characteristics of hereditary disease and demonstrate potential to identify mutations that are specific to a breed or kennel. Through pedigree analysis, fourteen samples were selected for genome sequencing, and they represented Golden Retrievers, Siberian Huskies, Standard Schnauzers, and Dalmatians. Following genome sequencing, orthologs of human genes are being investigated for comparison to human hereditary breast cancer sequencing data that were generated in our laboratory. This comparative genomics aspect may lead to the discovery of novel genes that influence breast cancer risk.

Citation Format: Anna L.W. Huskey, Carlos Lloveras-Fuentes, Katie Goebel, Nancy D. Merner. Canine pedigree analysis as a model of hereditary breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A62.
Abstract B18: ADSL controls pyrimidine metabolism and triple-negative breast tumorigenesis
Authors: Zurlo Giada , Simon Jeremy , Fan Cheng , Robinson Adam , Martinez Javier Rodriguez , Kriegsheim Alex , Locasale Jason , Perou Charles , Zhang Qing
Published: August 2018

Triple-negative breast cancer (TNBC) represents 15% of all breast carcinomas. The unfavorable prognosis and aggressive biology of TNBC highlight the need to find new targeted therapies. Previous study from our lab has shown that the prolyl hydroxylase EglN2 contributes to TNBC progression, with its depletion leading to a decrease in TNBC cell invasion. However, the underlying mechanism remains elusive. In our current study, we developed a novel enzyme-substrate trapping strategy followed by mass spectrometry and identified adenylosuccinate lyase (ADSL) as a potential EglN2 hydroxylase substrate. EglN2 binds directly with ADSL and catalyzes ADSL hydroxylation on proline residues, which might affect ADSL activity. We demonstrated that ADSL is specifically upregulated in TNBC cell lines and patients. Functionally, depletion of ADSL led to decreased anchorage-independent growth, TNBC cell invasion, and orthotopic tumor growth. Mechanistically, integrated analyses of metabolomics and RNA-seq showed that ADSL controls pyrimidine biosynthesis that is essential for TNBC cell proliferation and invasion. Therefore, our study suggests that EglN2-ADSL signaling axis can serve as a novel therapeutic avenue in TNBC by regulating pyrimidine metabolism.

Citation Format: Giada Zurlo, Jeremy Simon, Cheng Fan, Adam Robinson, Javier Rodriguez Martinez, Alex Kriegsheim, Jason Locasale, Charles Perou, Qing Zhang. ADSL controls pyrimidine metabolism and triple-negative breast tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B18.
Abstract B36: Progesterone receptor signaling in estrogen receptor-positive breast cancer
Authors: Young Amy , Guan Jane , Daemen Anneleen , Friedman Lori , Lin Kui
Published: August 2018

Approximately 65% of breast cancers are estrogen receptor alpha (ERα)-positive and depend on ERα signaling for growth. Clinical data indicate that ERα-positive breast cancers have a better prognosis when another hormone receptor, the progesterone receptor (PR), is coexpressed. Recent studies show that in the presence of its ligand progesterone, PR redirects ERα chromatin binding and transcriptional activity, thereby impacting prognosis and therapeutic response.

To further examine PR function, we knocked out the PGR gene in the ERα-positive breast cancer cell lines MCF-7 and T47D using CRISPR-mediated gene editing. Knockout clones from each model were analyzed for ER- and PR-mediated transcriptional activity and gene expression profile, as well as response to selective estrogen receptor degraders (SERDs).

The T47D cell line expresses high levels of PR in the presence or absence of estrogen, and stimulation with progesterone induces robust PR phosphorylation and modulation of PR target gene expression. Furthermore, progesterone attenuates estradiol-induced cell proliferation in a dose-dependent manner. Knockout of PR in this model abrogates the ability of progesterone to induce gene expression changes or attenuate estradiol-induced cell proliferation. In contrast, the MCF-7 cell line expresses low basal levels of PR, and is refractory to stimulation with progesterone. Interestingly, in estrogenic growth media, knockout of PR in this model results in basal gene expression changes that resemble a gene signature associated with tamoxifen resistance. Consistent with these findings, knockout of PR reduces the response to SERDs in a 5-day cell proliferation assay. ChIP-seq and RNA-seq analyses are ongoing to examine how loss of PR impacts ERα chromatin binding and transcriptional output.

Citation Format: Amy Young, Jane Guan, Anneleen Daemen, Lori Friedman, Kui Lin. Progesterone receptor signaling in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B36.
Abstract B66: The conditional requirement of HER3 in HER2-amplified breast cancers
Authors: Ruiz-Saenz Ana , Dreyer Courtney , Steri Veronica , Campbell Marcia , Gulizia Nathaniel , Moasser Mark
Published: August 2018

The amplification and overexpression of HER2 underlies the pathogenesis of a large subset of breast, gastric, and esophageal cancers as well as smaller subsets of many other types of cancer. A substantial body of work has confirmed that overactive HER2 signaling is etiologically linked with the pathogenesis, progression and metastasis of these cancers.

HER3, the preferred heterodimerization partner of HER2, is known to be critical for HER2-amplified tumorigenesis, and this has been thought to be due to its unique ability to activate PI3K/Akt pathway. This has fueled efforts to develop HER3 targeting agents to combine with HER2 inhibitors for the treatments of these cancers, and many such agents are in the preclinical and clinical pipelines.

We have been modeling HER2-HER3 signaling co-targeting in experimental models to foresee the activities and limitations of this clinical strategy and have identified potentially significant escape mechanisms that limit the therapeutic potential of this approach. This appears to be largely due to the mistaken attribution of PI3K/Akt signaling to HER3 and the dogma that HER2 cannot activate PI3K by itself. The functions of HER3 are not entirely understood and may be partly redundant with HER2, enabling HER2 to escape the requirement for HER3. In this regard, we found that HER3-knock out HER2-overexpressing cells form tumors, and some of these tumors exhibit increased HER2 phosphorylation and Akt activation. We validated, in three different HER2-amplified cancer cell lines, that phosphorylated HER2 drives PI3K-mediated Akt activation in a HER3-independent manner. Interestingly, we found that HER2 interacts with the regulatory subunit of PI3K in these cells. We characterized this interaction and identified a tyrosine in the C-terminal tail of HER2 involved in PI3K binding. Most importantly, in vivo work demonstrates that mutation of this tyrosine reduces the transforming and tumorigenic potential of HER2-amplified cancer cells.

This work highlights that the activation of PI3K/Akt signaling by HER3 may be an important but not essential function and may ultimately be redundant with the functional capabilities of massively overexpressed HER2. Therefore, building a better understanding of the molecular mechanisms that underlie HER2-HER3-induced tumorigenesis is essential for the development of more effective therapeutic strategies.

Citation Format: Ana Ruiz-Saenz, Courtney Dreyer, Veronica Steri, Marcia Campbell, Nathaniel Gulizia, Mark Moasser. The conditional requirement of HER3 in HER2-amplified breast cancers [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B66.
Abstract IA15: PARP inhibitor resistance and acquired vulnerability in breast cancer
Authors: D’Andrea Alan D.
Published: August 2018

Large-scale genomic studies have demonstrated that some breast cancers, especially triple-negative breast cancers (TNBCs), harbor genetic and epigenetic alterations in homologous recombination repair (HRR) pathway genes. The most commonly altered HRR genes are BRCA1 and BRCA2, followed by other Fanconi anemia genes including FANCN/PALB2, FANCO/RAD51, FANCJ/BRIP, and FANCA. Loss of HRR causes genomic instability, hyperdependence on alternative DNA repair mechanisms, and enhanced sensitivity to platinum analogues, topoisomerase inhibitors, and PARP inhibitors (PARPi). The synthetic lethal interaction with PARPi is being exploited therapeutically in diverse clinical contexts and most notably in ovarian cancer where the PARPi olaparib is FDA approved for use in patients with germline BRCA1/2 mutations. PARP inhibitor resistance has already emerged as a vexing clinical problem for the treatment of BRCA1/2 deficient tumors. The most prevalent mechanism of PARPi resistance is secondary events that cancel the original HRR alteration and restore HRR proficiency. However, PARPi resistance may still develop without restoration of HRR proficiency via disruption of multiple proteins, such as PTIP or CHD4, that leads to replication fork (RF) stabilization. Importantly, this latter mechanism—namely, the restoration of RF stability—appears to be a highly prevalent mechanism of PARP inhibitor resistance in vitro and in vivo, particularly in tumor cells with an underlying BRCA2 deficiency. Due to their underlying deficiency in BRCA2 and inability to generate RAD51 nucleofilaments, these tumor cells are unable to restore HRR mechanisms. Instead, these cells acquire PARP inhibitor resistance by limiting the nucleolytic degradation of their stalled replication forks. We have recently made the surprising observation that BRCA2-deficient tumors can become resistant to PARPi by downregulating the expression of the polycomb repressive complex PRC2, a methyltransferase complex containing EZH2, SUZ12, EED, and RbAp48. Importantly, downregulation of PRC2 results in the reduced recruitment of the nuclease MUS81 to the RF, thereby providing a novel mechanism of RF protection and PARPi resistance. A molecular understanding of PARP inhibitor resistance mechanisms may allow the generation of a new class of drugs, or a repurposing of existing drugs, which may reverse this resistance and extend the use of PARP inhibitors to more tumor types.

Citation Format: Alan D. D’Andrea. PARP inhibitor resistance and acquired vulnerability in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr IA15.
Abstract 13: PRMT1 as a therapeutic target in diffuse large B-cell lymphoma
Authors: Goverdhan Aarthi , Lee Heng-Huan , Havranek Ondrej , Davis Richard Eric , Hung Mien-Chie
Published: August 2018

Diffuse large B-cell lymphomas (DLBCL) harbor recurrent abnormalities in epigenetic regulators, such as loss-of-function mutations in EP300 and CREBBP. These include gain-of-function mutations in EZH2, largely restricted to the germinal center B-Cell (GCB) subtype. An EZH2 inhibitor EPZ-6438 showed efficacy as a single agent in phase I clinical trials in relapsed/refractory GCB-DLBCL. We examined the effect of EZH2 inhibition in GCB-DLBCL cell lines, and found an increase in histone arginine methylation in surviving cells, which was attributable to increased Type I PRMT activity. Specifically, we discovered that EZH2 inhibitors activated PRMT1, which mediated protective effects through transcriptional changes at survival genes such as BCL2, and through modulation of B-cell receptor signaling. This exciting observation suggested an opportunity for combination therapy with PRMT1 inhibitors. Since PRMT1 is poorly studied in DLBCL, we performed single-agent studies in lymphoma cell lines and discovered that inhibition of PRMT1 elicited remarkable suppression of lymphoma cell growth and viability. Analysis of gene expression data from patients indicates that PRMT1 is overexpressed in untreated DLBCL tumor samples, compared to normal B-lymphocytes. Additionally, survival analysis revealed that patients with high PRMT1 expression showed poor overall survival. These observations support the potential for combinatorial treatment with EZH2 and PRMT1 inhibitors in patients. To summarize, our studies uncover a novel epigenetic target in DLBCL, and offer avenues for improving the antitumor efficacy of EZH2 inhibitors.

Citation Format: Aarthi Goverdhan, Heng-Huan Lee, Ondrej Havranek, Richard Eric Davis, Mien-Chie Hung. PRMT1 as a therapeutic target in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 13.
Abstract IA22: Targeting CARs to the TRAC locus enhances T-cell potency
Authors: Sadelain Michel , Eyquem Justin
Published: August 2018

Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumor rejection. The most successful CARs used to date are those targeting CD19, which offer the prospect of complete remissions in patients with chemorefractory/relapsed B cell malignancies. CARs are typically transduced into patient T cells using γ-retroviral or other randomly-integrating retroviral vectors (RVs), which may result in variegated CAR expression and transcriptional silencing. Recent advances in genome editing enable efficient sequence-specific modifications in human primary cells, including site-specific transgene integration into the CCR5 and AAVS1 loci in T lymphocytes. Using CRISPR/Cas9 to edit human T cells, we have established conditions yielding efficient target gene disruption and CAR insertion in a single step. We have found that directing a CD19 CAR to the human T-cell receptor (TCR) alpha chain (TRAC) locus not only results in efficient and uniform CAR expression in human peripheral blood T cells, but, remarkably, also enhances T-cell potency, vastly outperforming that of conventionally generated CAR T cells. We further show that CAR gene expression under the control of the TCR alpha promoter minimizes tonic signaling and allows effective regulation of CAR expression. These findings provide important insights into CAR immunobiology and highlight the potential of CRISPR/Cas9 genome editing to advance immunotherapies.

Citation Format: Michel Sadelain, Justin Eyquem. Targeting CARs to the TRAC locus enhances T-cell potency [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA22.
Abstract A25: Tissue transglutaminase interacts with Frizzled 7 in ovarian cancer stem cells
Authors: Condello Salvatore , Sima Livia , Ivan Cristina , Cardenas Horacio , Schiltz Gary , Mishra Rama K , Matei Daniela
Published: August 2018

Increasing evidence correlates the metastatic progression and post-chemotherapy recurrence of ovarian cancer (OC) to a small population of cancer stem cells (CSCs), resistant to traditional chemotherapy and responsible for tumor relapse. Tissue transglutaminase (TG2) is a multifunctional protein, with enzymatic and scaffold functions, overexpressed in ovarian carcinomas, where it promotes metastasis. Independently of its enzymatic function, TG2 can be secreted in the extracellular compartment where it increases cell adhesion to the fibronectin (FN) matrix by regulating integrins clustering and matrix reorganization. We hypothesized that by binding to the integrins and FN, TG2 promotes survival of CSCs and supports OC spheroid formation. Our data demonstrate that TG2, β1 integrin, and FN1 expression are markedly increased in OC spheroids compared with monolayers and are enriched in successive sphere generations. Immunofluorescence (IF) staining supports that TG2 forms abundant complexes with β1 integrin and FN in cells grown as spheroids. TG2 expression is strongly correlated with β1 integrin (ITGB1) (R=0.23, P<0.0001) and with FN1 (R=0.39, P<0.0001) in the TCGA ovarian cancer database, which houses gene expression data of 560 clinically annotated HGSOC tumors. Patients with high TG2 and ITGB1 expression levels had an increased estimated risk of death when compared to those with low TG2 and ITGB1 expression levels (P=0.00652). Use of function-inhibiting antibody against the FN-binding domain of TG2 (4G3) dramatically suppressed TG2/FN/β1 integrin complex formation, spheroid proliferation, and tumor growth in a xenograft model. Furthermore, disruption of TG2/FN complex formation altered key oncogenic signaling pathways, and in particular blocked the canonical Wnt/β-catenin signaling pathway essential to sustaining cancer cell stemness. The Wnt receptor Frizzled genes (Fzd1, Fzd7 and Fzd8) showed the greatest difference in gene expression, indicating a direct correlation between TG2/FN complex and Wnt pathway activation. By co-immunoprecipitation and IF staining we demonstrated that TG2 colocalizes and directly binds to Fzd7 receptor. In conclusion, our data support that TG2/FN/β1 integrin complex formation is essential to promoting ovarian CSCs survival by acting as coreceptor complex with Fzd7 in regulating β-catenin signaling and represents a new target to eradicate chemotherapy-resistant tumors.

Citation Format: Salvatore Condello, Livia Sima, Cristina Ivan, Horacio Cardenas, Gary Schiltz, Rama K Mishra, Daniela Matei. Tissue transglutaminase interacts with Frizzled 7 in ovarian cancer stem cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A25.
Abstract A28: Beta-escin inhibits ovarian cancer metastasis by targeting the tumor microenvironment
Authors: Kenny Hilary A. , Lal Madhu , Shen Min , Kara Betul , Chiang Chun-Yi , Watters Karen , Hart Peter , Ferrer Marc , Lengyel Ernst
Published: August 2018

The high mortality rate that results from ovarian cancer (OvCa) is caused by the wide dissemination of cancer cells within the abdominal cavity. OvCa cells metastasize to the peritoneum, which is covered by a single layer of mesothelial cells, and invade into the underlying stroma, composed of extracellular matrices including fibronectin (FN) and stromal cells such as fibroblasts. Using a three-dimensional organotypic quantitative high-throughput screening platform (3D-qHTS) modeling the peritoneum, we discovered that beta-escin inhibited OvCa adhesion and invasion to the peritoneum. Beta-escin is a natural triterpenoid saponin from Chinese horse chestnut seeds and has a variety of known pharmacologic effects. We hypothesize that beta-escin targets both the cancer cells and the stromal cells to inhibit the metastatic niche. The therapeutic effects of beta-escin and horse chestnut seed extract were tested in vivo using xenograft and syngeneic models of OvCa prevention and intervention. The mechanisms of beta-escin action in cancer cells and microenvironmental cells was explored in vitro using OvCa cell lines and primary human mesothelial cells and fibroblasts, and in vivo using different mouse models of metastasis. Moreover, a collection of 160 analogs of beta-escin were gathered and screened using our 3D-qHTS platform for inhibitory activity in OvCa cell adhesion/invasion. Our results reveal that beta-escin and horse chestnut extract taken orally inhibit metastasis in both OvCa prevention and intervention models. In addition, beta-escin mechanistically depresses the pluripotent stem cell population, inflammatory cytokine secretion, HIF-1alpha expression, and fibronectin production, while it increases autophagy in the tumor microenvironment. Furthermore, three distant analogs of beta-escin inhibited OvCa metastasis, and all three are cardiac glycosides. Taken together, we reveal a potential therapeutic value for beta-escin and/or analogs of beta-escin in preventing and treating OvCa dissemination.

Citation Format: Hilary A. Kenny, Madhu Lal, Min Shen, Betul Kara, Chun-Yi Chiang, Karen Watters, Peter Hart, Marc Ferrer, Ernst Lengyel. Beta-escin inhibits ovarian cancer metastasis by targeting the tumor microenvironment. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A28.
Abstract A52: Potassium channel activator minoxidil (Rogaine) as a novel single-agent or combination therapy in ovarian cancer
Authors: Fukushiro-Lopes Daniela , Jain Megan , Khalid Maryam , Hegel Alexandra , Gentile Saverio
Published: August 2018

Recent advances in cancer therapy treatments for ovarian cancer have been severely hindered by limited drug availability, excessive side effects, and drug resistance and the difficulty in finding and targeting proteins governing important events in ovarian cancer cells. Novel therapeutic agents targeting specific molecular pathways involved in ovarian cancer are urgently needed.

During our investigation to identify new and critical targets in cancer, we found a novel role for the potassium ion channel Kir6.2 in ovarian cancer cells. Our bioinformatics investigation showed that Kir6.2 is expressed in ovarian cancers. We discovered that stimulation of the Kir6.2 channel activity with the small molecule minoxidil (Rogaine®) produces a strong inhibition on ovarian cancer cells’ growth. However, minoxidil had no significant effects on cells that do not express Kir6.2.

We describe a biochemical signaling linking activation of Kir6.2 potassium channel to the tumor suppressor mechanism, which includes a Ca2+-dependent production of superoxide species (ROS), which leads to a significant increase of DNA damage. Consequently, cells die upon activation of apoptosis via caspase-3. Minoxidil is an FDA-approved drug that presents very limited side effects. Therefore, our data are important because for the first time they reveal that use of the Kir6.2 potassium channel activator, e.g., Rogaine®, could be considered as a potential single-agent novel treatment for ovarian cancer.

Clinicians’ approaches for treating advanced ovarian cancer patients are strongly limited by the concern that current chemotherapeutic agent will raise toxicity. For example, although genotoxic chemotherapeutic agents such as doxorubicin or platinum-containing molecules of carboplatin are among the most effective drug classes in ovarian cancer, dose-dependent toxicity of these agents is often the cause for interrupting the treatment.

Our experiments demonstrated that the Kir6.2 activator minoxidil potentiates the antineoplastic effects of doxorubicin as well as of carboplatin by exacerbating their DNA-damaging effects. This discovery is important because it offers the opportunity to design a clinically effective therapeutic strategy in which minoxidil could be administered in combination with genotoxic agents to limit their toxicity in ovarian cancer patients.

Citation Format: Daniela Fukushiro-Lopes, Megan Jain, Maryam Khalid, Alexandra Hegel, Saverio Gentile. Potassium channel activator minoxidil (Rogaine) as a novel single-agent or combination therapy in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A52.
Abstract A66: The tissue transglutaminase 2–fibronectin protein complex: A new target in ovarian cancer
Authors: Sima Livia Elena , Yakubov Bakhtiyor , Condello Salvatore , Cardenas Horacio , Zhang Zhong Yin , Chen Lan , Matei Daniela Elena
Published: August 2018

Here we describe a small-molecule inhibitor (SMI) that disrupts the tissue transglutaminase 2 (TG2)-fibronectin (FN) interaction. TG2 is a multifunctional protein, with enzymatic (transglutaminase, isopeptidase, protein disulfide isomerase), GTPase, and extracellular matrix (ECM) scaffold activities. TG2 directly interacts with FN through its N-terminus domain, playing an important role in stabilizing ternary TG2/FN/integrin complexes, which regulate cell adhesion to the matrix. Our group has previously shown that TG2 overexpressed in ovarian cancer (OC) promotes epithelial-to-mesenchymal transition (EMT) contributing to OC cell invasiveness and metastatic behavior. We showed that the interaction with FN is critical to these functions. Proximity ligation assay was used to measure TG2/integrin beta 1 interaction in OC tumors and identified TG2/integrin beta 1 complex in >50% of ovarian tumors, irrespective of histologic type. Based on those findings, we conducted the high-throughput screening and identified TG53 as the lead TG2/FN inhibitory compound. Structure-activity relationship-based approach was used to optimize the physicochemical properties of TG53, leading to design and chemical synthesis of 6 analogues (MT1-6). All analogues blocked TG2/FN interaction, as measured by an ELISA assay, and OC cell adhesion to FN, as measured by a solid phase assay, MT4 being the most potent inhibitor. MT4 downregulates integrin-initiated signaling in OC cells, leading to downstream inhibition of FAK and ERK. Immunofluorescence staining demonstrated that the actin cytoskeleton adopts a cortical localization pattern in MT4-treated cells. MT4 caused membrane ruffling delaying the formation of stable focal contacts and mature adhesions points. Pretreatment with MT4 sensitized OC cells to paclitaxel, as measured by colony-forming assay. In an intraperitoneal OC mouse model, MT4 caused decreased OC cell adhesion to the peritoneum, inhibiting peritoneal dissemination. Hence, MT4 is a potential candidate to be developed and used in combination with current chemotherapeutics to target OC tumor progression. Further studies will continue to optimize this new class of TG2/FN SMIs for potential clinical use in OC.

Citation Format: Livia Elena Sima, Bakhtiyor Yakubov, Salvatore Condello, Horacio Cardenas, Zhong Yin Zhang, Lan Chen, Daniela Elena Matei. The tissue transglutaminase 2–fibronectin protein complex: A new target in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A66.
Abstract B23: The evolution of estrogen receptor signaling in the progression of endometriosis to endometriosis-associated ovarian cancer
Authors: Boisen Michelle , Andersen Courtney , Sikora Matt , Ma Tianzhou , Tseng George , Vlad Anda , Elishaev Esther , Chandran Uma , Edwards Robert , Oesterreich Steffi
Published: August 2018

Objectives: To investigate changes in estrogen receptor–alpha (ERα) signaling during the progression of endometriosis to endometriosis-associated ovarian cancer (EAOC) as a putative driver of malignant transformation.

Methods: We procured tissue samples of normal endometrium (n=23), endometriosis (benign, n=19; atypical, n=11; concurrent with EAOC, n=9), and EAOC (n=21). In this cohort, we evaluated expression of a 236-gene signature of estrogen signaling. ANOVA and unsupervised clustering were used to identify distinct gene expression profiles across disease states. These profiles were compared to public profiles of estrogen regulation in preclinical cancer models from the Gene Expression Omnibus (GEO). Gene set enrichment analysis (GSEA) was performed to determine whether gene expression in EAOC was consistent with ERα activity.

Results: ANOVA revealed 158 differentially expressed genes (q<0.05) and unsupervised clustering identified 4 distinct gene clusters. Cluster 1 comprises genes with increasing expression from benign endometriosis to EAOC (e.g. FGF18, ESR2). Clusters 2 and 3 include genes that are overexpressed or downregulated in EAOC compared to benign endometriosis, respectively (e.g., NRIP1, IGFBP3). Cluster 4 consists of genes with an incremental decrease in expression from benign endometriosis to EAOC (e.g., ESR1, PGR, and GREB1). The estrogen signaling profile of EAOC was not consistent with activated ERα in the preclinical models. Further, GSEA did not identify signatures of activated ERα in EAOC but instead identified expression patterns consistent with loss of ERα function and development of endocrine resistance.

Conclusions: Gene expression data suggest that classical ERα signaling becomes inactivated throughout the progression of endometriosis to EAOC. Rather, the gene expression pattern in EAOC is more consistent with profiles of endocrine resistance. De-repression of ERα target genes such as FGF18 may contribute to evolution of endometriosis into EAOC.

Citation Format: Michelle Boisen, Courtney Andersen, Matt Sikora, Tianzhou Ma, George Tseng, Anda Vlad, Esther Elishaev, Uma Chandran, Robert Edwards, Steffi Oesterreich. The evolution of estrogen receptor signaling in the progression of endometriosis to endometriosis-associated ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B23.
Abstract B64: The novel ZIP4 regulation and its role in cancer stem cell-related activities in ovarian cancer
Authors: Xu Yan , Fan Qipeng , Cai Qingchun , Nephew Kenneth P.
Published: August 2018

The role and regulation of ZIP4, a zinc (Zn) transporter, have been studied in the context of extracellular Zn transporting. Factors other than Zn with ZIP4 regulatory effects are essentially unknown. In addition, its role in cancer stem cell (CSC) has not been reported in any type of cancer. RNA-seq analyses were conducted to compare genes differentially expressed in less and more aggressive epithelial ovarian cancer (EOC) cells. ZIP4 expression and its regulation was assessed by immunoblotting, quantitative PCR, or immunofluorescence staining in human ovarian tissues. CSC-related activities were examined to evaluate the role of ZIP4 in human high-grade serous ovarian cancer (HGSOC) cells. RNAi and CRISPR techniques were used to knock down or knock out ZIP4 and related genes. EOC tumors overexpressed ZIP4 when compared with normal and benign tissues. ZIP4 knockout significantly reduced several CSC-related activities in EOC cells, including proliferation, anoikis resistance, colony formation, spheroid formation, drug resistance, and side-population (SP). Only ZIP4-expressing SP highly expressed known CSC markers ALDH1 and OCT4. ZIP4 knockout dramatically reduced tumorigenesis and ZIP4 overexpression increased tumorigenesis in vivo. In addition, the ZIP4-expressing SP population had the tumor initiating activity. Moreover, the oncolipid lysophosphatic acid (LPA) effectively upregulated ZIP4 expression via the nuclear receptor peroxisome proliferator-activated receptor gamma, and LPA’s promoting effects in CSC-related activities in HGSOC cells was at least partially mediated by ZIP4 in an extracellular Zn-independent manner. Our critical data imply that ZIP4 is a new and important cancer stem cell regulator in EOC. Our data also provide an innovative interpretation for the apparent disconnection between low levels of zinc and upregulation of ZIP4 in EOC tissues.

Citation Format: Yan Xu, Qipeng Fan, Qingchun Cai, Kenneth P. Nephew. The novel ZIP4 regulation and its role in cancer stem cell-related activities in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B64.
Abstract B67: Antitumor effect of black tea pigments, theaflavin-3/3’-gallate, against cisplatin-resistant ovarian cancer cells
Authors: Chen Yi Charlie
Published: August 2018

Ovarian cancer ranks fifth in cancer deaths among women in the United States, accounting for approximately 5% of all cancer deaths. Acquired resistance to conventional platinum-based chemotherapy drives the development of more selective anticancer drugs. Theaflavin-3-gallate (TF2a) and theaflavin-3’-gallate (TF2b) are the major bioactive components in black tea. In this study, we evaluated TF2a and TF2b as potential anticancer agents using human ovarian cancer cells. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37˚C in a humidified incubator with 5% CO2. The [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, flow cytometry, and Western blot analysis were used to confirm that TF2a and TF2b exhibited a potent growth-inhibitory effect against cisplatin-resistant ovarian cancer A2780/CP70 cells. Treatment with TF2a and TF2b (10-40 μM) significantly decreased A2780/CP70 cell viability (p< 0.05). Treatment with TF2a and TF2b (5, 10 and 20 μM) increased the percentage of apoptotic cells in a concentration-dependent manner. Western blot analysis was used to evaluate the expression of proteins related to apoptosis in A2780/CP70 cells treated with designated concentrations (0, 5, 10, and 20 μM) of TF2a and TF2b for 24 h. The protein levels of procaspase-3/7 for TF2a-treated cells and procaspase 3 for TF2b-treated cells decreased significantly in A2780/CP70 cells (p< 0.05). Treatment with TF2a or TF2b significantly increased the protein levels of cleaved caspase-3/7, full-length PARP, and cleaved PARP in A2780/CP70 cells. Our study demonstrated that TF2a and TF2b are potential agents for treating platinum-resistant ovarian cancer by inducing apoptosis.

Note: This abstract was not presented at the conference.

Citation Format: Yi Charlie Chen. Antitumor effect of black tea pigments, theaflavin-3/3’-gallate, against cisplatin-resistant ovarian cancer cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B67.
Abstract IA04: Mitotic progression connects DNA damage to activation of antitumor immune responses
Authors: Greenberg Roger A.
Published: August 2018

Inflammatory gene expression following genotoxic cancer therapy is well documented, yet the events underlying its induction remain poorly understood. Inflammatory cytokines modify the tumor microenvironment by recruiting immune cells and are critical for both local and systemic (abscopal) tumor responses to radiotherapy. An enigmatic feature of this phenomenon is its delayed onset (days), in contrast to the acute DNA damage responses that occur in minutes to hours. Such dichotomous kinetics implicate additional rate limiting steps that are essential for DNA-damage induced inflammation. Here, we show that cell cycle progression through mitosis following DNA double-strand breaks (DSBs) leads to the formation of micronuclei, which precede activation of inflammatory signaling and are a repository for the pattern recognition receptor cGAS. Inhibiting progression through mitosis or loss of pattern recognition by cGAS-STING impaired interferon signaling and prevented the regression of abscopal tumors in the context of ionizing radiation and immune checkpoint blockade in vivo. These findings implicate temporal modulation of the cell cycle as an important consideration in the context of therapeutic strategies that combine genotoxic agents with immune checkpoint blockade.

Citation Format: Roger A. Greenberg. Mitotic progression connects DNA damage to activation of antitumor immune responses. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr IA04.
Abstract A02: Succinate dehydrogenase deficiency in a PDGFRA mutated gastrointestinal stromal tumor
Authors: Belinsky Martin G. , Rink Lori , Cai Kathy K. , Luo Biao , Zhou Yan , Mehren Margaret von
Published: August 2018

Most gastrointestinal stromal tumors harbor mutually exclusive gain-of-function mutations in the receptor tyrosine kinase (RTK) KIT (70-80%) or in the related receptor PDGFRA (~10%). Mutations in PDGFRA are most commonly found in the TK domain encoded by exon 18, conferring resistance to approved tyrosine kinase inhibitors (TKI). A more recently defined subset of GIST exhibit deficiencies in the succinate dehydrogenase (SDH) enzyme complex as a result of genetic or epigenetic inactivation of one of the four SDH subunit genes (SDHA-D). SDH-deficient GISTs generally lack mutations in KIT and PDGFRA and do not respond to imatinib (IM), although these GISTs may respond to TKIs targeting VEGFR, such as sunitinib and regorafenib. Whole-exome sequencing (WES) of two GISTs from a single case carrying the IM-resistant exon 18 PDGFRA mutation D842V was carried out to identify additional potential targets for therapy. Surprisingly, a somatic mutation in exon 4 of the SDHB subunit gene (c.291_292delCT, p.I97Mfs*21) was identified in both tumors. Sanger sequencing confirmed the presence of this inactivating mutation, and immunohistochemistry for the SDHB subunit demonstrated that these tumors were in fact SDH deficient. IHC for the SDHB subunit across a panel of ~100 GIST cases failed to detect SDH deficiency in other GISTs with RTK mutations. To our knowledge this is the first reported case of an SDH-deficient GIST harboring a gain-of-function mutation in the PDGFRA receptor, and this finding has implications for the molecular-based classification and treatment of GIST.

Citation Format: Martin G. Belinsky, Lori Rink, Kathy K. Cai, Biao Luo, Yan Zhou, Margaret von Mehren. Succinate dehydrogenase deficiency in a PDGFRA mutated gastrointestinal stromal tumor [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A02.
Abstract A26: Pimozide, an antipsychotic derivative, targets the STAT signaling pathway in osteosarcoma
Authors: Angulo Pablo , Chastain Katherine , Subramaniam Dharmalingam , Anant Shrikant
Published: August 2018

Objective: Osteosarcoma (OS) is the most common pediatric bone cancer worldwide with an annual US incidence of 800. Chemotherapy and surgery have resulted in a marginal increase in survival of localized OS. However, that same treatment regimen has not been as successful for improving metastatic OS, as it yields an abysmal 5-year overall survival of less than 30% in patients afflicted with the disease. There have not been new successful standardized treatment agents in the last 2 decades for OS. Current standard practice still implements the same chemotherapeutic agents: cisplatin, doxorubicin, and methotrexate. These nonspecific agents have failed to yield optimal survival in metastatic OS patients. Hence, there is a need to utilize different agents that can specifically target signaling pathways involved in the pathogenesis of OS. One such pathway that has been found to be instrumental in the pathogenesis of OS is the JAK/STAT pathway, and the antipsychotic medication pimozide appears to be able to halt OS growth through inhibiting this pathway. Herein, we investigated the effects of pimozide on OS cancer growth and elucidated its mechanistic action through STAT signaling pathway.

Methods: Human OS cell lines KHOS, MG63, and SJSA cell lines and normal osteoblast cells were used in the study. The effects of pimozide on cell proliferation were assessed by hexosaminidase and clonogenicity assays. Effects of pimozide on stem cells were evaluated by colonosphere formation. Furthermore, the effect of pimozide on OS cells was evaluated by live cell fluorescent staining. Western blot was utilized to evaluate the effect on STAT protein expression.

Results: Pimozide treatment resulted in a dose- and time-dependent inhibition of proliferation and colony formation in all three osteosarcoma cell lines but not in osteoblast cells. It induces caspase 3 and 7 activity. Pimozide also showed reduced live cell fluorescent staining. Pimozide significantly reduced the number and size of spheroid formation. In addition, pimozide inhibits OS stem cell marker protein CD44 expression. Moreover, pimozide treatment decreased the expression of STAT5 signaling proteins on Western blot, which suggests that pimozide inhibits OS growth through STAT signaling pathway.

Conclusion: Together, these data suggest that pimozide inhibits OS cancer growth by inhibiting the STAT signaling pathway. Pimozide could serve as a potential therapeutic agent for OS.

Citation Format: Pablo Angulo, Katherine Chastain, Dharmalingam Subramaniam, Shrikant Anant. Pimozide, an antipsychotic derivative, targets the STAT signaling pathway in osteosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A26.
Abstract B09: Novel sarcoma treatments identified via drug sensitivity/resistance screening of sarcoma cell lines
Authors: Grad Iwona , Ayuda Pilar , Hanes Robert , Enserink Jorrit , Myklebost Ola
Published: August 2018

Sarcomas comprise a plethora of subtypes and each should be regarded as ultra-orphan malignancy. Over the last century incremental improvements of sarcoma therapies have provided better survival; however, treatment options appear to have been exhausted and new alternatives beyond traditional chemotherapeutic regimens are much needed. To broaden the repertoire of antisarcoma drugs, we applied a direct drug testing approach. Using a library of 349 anticancer compounds (FDA-approved/under clinical trial) and panels of liposarcoma and osteosarcoma cell lines, we selected a group of potential novel antisarcoma therapeutics and retested them against cells from primary tumors. We are also investigating genomic and molecular features to find potential drug response biomarkers. Since all those compounds have been approved or are under clinical trials, they present a unique shortcut repurposing opportunity for personalized sarcoma treatment.

Citation Format: Iwona Grad, Pilar Ayuda, Robert Hanes, Jorrit Enserink, Ola Myklebost. Novel sarcoma treatments identified via drug sensitivity/resistance screening of sarcoma cell lines [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B09.
Abstract B22: Uncovering the role of lysyl oxidase-like 2 in rhabdomyosarcoma
Authors: Rabaiget Olga Almacellas , Martínez Juan Huertas , Monclús Silvia Garcia , Varona Santiago Rello , Martín Herrero , Alemany Roser López , Tirado Oscar M.
Published: August 2018

Rhabdomyosarcoma (RMS) is the most common soft-tissue malignancy in childhood and adolescence that originates as a consequence of regulatory disruption in the growth and differentiation of muscle precursor cells. Members of the lysyl oxidase family of proteins (LOX and LOXL1-4) are amine oxidases that catalyze the covalent crosslinking of collagen and elastin in the extracellular matrix. Apart from their traditional role, novel functions have been attributed to these proteins related to tumor progression. In this study we sought to unravel the potential role of this family of proteins during the progression of RMS. By qRT-PCR and Western blot, lysyl oxidase-like 2 (LOXL2) was found overexpressed in RMS cell lines compared to other sarcoma cells. Then we characterized LOXL2 in RMS cell lines by analyzing the subcellular localization by immunofluorescence and subcellular fractionation. Post-translational modifications were also analyzed: glycosylation and proteolytic processing by tunicamycin treatment and secretion by analysis of the conditioned media by Western blot. Moreover, we characterized the neoplastic phenotype after silencing LOXL2 in two RMS cell lines (RH4 and CW9019). Stable LOXL2 knockdown resulted in a decrease in their clonogenic, proliferative, and cell migratory capabilities. On the other hand, reexpression of LOXL2 in RH28 cells using wild-type or mutated (catalytically inactive) constructs resulted in an increase in cell proliferation and migration, independently of its catalytic activity. Finally, in vivo experiments using an orthotopic model of tumor generation inside the muscle will be carried out to further evaluate the tumorigenic role of LOXL2. Taking all into consideration, our results suggest an oncogenic role of LOXL2 in RMS.

Citation Format: Olga Almacellas Rabaiget, Juan Huertas Martínez, Silvia Garcia Monclús, Santiago Rello Varona, David Herrero Martín, Roser López Alemany, Oscar M. Tirado. Uncovering the role of lysyl oxidase-like 2 in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B22.