bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3401: Role of membrane-associated guanylate kinase inverted 2 in advanced and castration-resistant prostate cancers
Authors: Le Bao , Hammer Kimberly , Frantskevich Katya , Ong Irene , Huang Wei , Marker Paul
Published: July 2017

Background: Prostate cancer is the most common non-cutaneous malignancy for men in the U.S. Patients with advanced and/or metastatic prostate cancer are commonly treated with androgen deprivation therapy. Unfortunately, incurable castration resistant prostate cancer commonly develops 2-3 years after initial treatment.

Methods: To identify candidate cancer genes involved in the development of castration resistance, the Sleeping Beauty (SB) transposon system and androgen-sensitive LNCaP cell line were used in a forward mutagenesis screen designed to model the transition from androgen sensitive to androgen insensitive prostate cancer. MAGI-2 was identified in the screen as a common insertion site gene, potentially involved in the transition to androgen independence. Clinical significance of MAGI-2 in prostate cancer was further supported by human TCGA data. Functional assays were used to confirm the involvement of MAGI-2 in prostate cancer progression.

Results: MAGI-2 expression was prognostic for patient survival outcome. Ectopic expression of MAGI-2 in 22Rv1 cells resulted in a decrease in cell proliferation and colony formation in in vitro studies. At the molecular level, overexpression of MAGI2 induced a suppression of known androgen responsive genes. MAGI-2 overexpression also limited the growth of grafts compared to empty vector grafts in in vivo xenograft models.

Conclusion: MAGI-2 was identified in a forward mutagenesis screen modeling the development of castration resistant prostate cancer. Human protein expression data from patient tissues supported a possible role for MAGI-2 in prostate cancer. Functional studies confirmed the potential involvement of MAGI-2 in prostate cancer progression. Future studies are seeking to elucidate the functional role and mechanism-of-action for MAGI-2 in prostate cancer. Ultimately, this may identify MAGI-2-regulated signaling pathways as therapeutic targets for prostate cancer.

Citation Format: Bao Le, Kimberly Hammer, Katya Frantskevich, Irene Ong, Wei Huang, Paul Marker. Role of membrane-associated guanylate kinase inverted 2 in advanced and castration-resistant prostate cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3401. doi:10.1158/1538-7445.AM2017-3401
Abstract 3403: Telomere shortening in pancreatic cancer is correlated to KRAS mutation
Authors: Matsuda Yoko , Izumiyama Naotaka , Fujiwara Mutsunori , Ishikawa Naoshi , Aida Junko , Takubo Kaiyo , Ishiwata Toshiyuki , Arai Tomio
Published: July 2017

Background: Pancreatic cancer is characterized by genomic complexity and chromosomal instability, and atypical mitotic figures are morphological features of this phenotype. Previously we have reported that approximately 30% of total mitosis in pancreatic cancer was atypical including multipolar, lag-type, ring and asymmetrical mitosis, and anaphase bridges, and the number of total mitosis and atypical mitosis in pancreatic cancers was correlated with aggressive phenotype and prognosis (Pancreatology, 2016). In the present study, we clarified the relation between atypical mitotic figures, telomere length, and genetic abnormality in the pancreatic cancer.

Methods: We surveyed the mitotic figures of the normal epithelium, pancreatic intraepithelial neoplasias (PanINs), and pancreatic cancers using surgically resected pancreatic cancer specimens (n=40). Telomere length was analyzed using quantitative fluorescent in situ hybridization technique. We also analyzed mutations of Kras codon 12 and 13 by PCR and microsatellite instability by immunohistochemical staining of MLH1, MSH2, MSH6 and PMS2.

Results: Pancreatic cancer and duodenal epithelium showed significantly higher mitotic indices as compared with the duct, acinar cells, and PanINs. Normal mitosis was also higher in pancreatic cancers and the duodenal epithelium, while atypical mitosis was significantly elevated only in pancreatic cancers. Number of total mitosis and atypical mitosis were negatively correlated with telomere length, suggesting that telomere shortening plays important roles in cancer proliferation and chromosomal instability. In comparison with normal ducts, telomere length was decreased in PanIN-1, -2 and -3 and cancer. Furthermore, telomere length was gradually shorter among PanIN grades. Most of pancreatic cancers harbored mutations in Kras codon 12, and pancreatic cancer cases with Kras mutation showed shorter telomere length as compared with cases without Kras mutation. All pancreatic cancer cases in the present study were microsatellite stable.

Conclusion: Our data strongly suggest that telomere shortening occurs in the early stages of pancreatic carcinogenesis and progresses with precancerous development. Telomere shortening and chromosomal instability in the duct epithelium plays key roles on carcinogenesis of the pancreas.

Citation Format: Yoko Matsuda, Naotaka Izumiyama, Mutsunori Fujiwara, Naoshi Ishikawa, Junko Aida, Kaiyo Takubo, Toshiyuki Ishiwata, Tomio Arai. Telomere shortening in pancreatic cancer is correlated to KRAS mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3403. doi:10.1158/1538-7445.AM2017-3403
Abstract 341: Investigating a novel GPCR ITIM motif in regulating Shp2 binding, signaling, and migration
Authors: Paulukinas Ryan D. , Belcastro Lili T. , Moore Catherine C.
Published: July 2017

Aberrant expression of CXCR4, a chemokine G protein-coupled receptor (GPCR), promotes a migratory and invasive phenotype during breast cancer metastasis, however the mechanism is unknown. Here we investigate the role of CXCR4 tyrosine phosphorylation within a novel ITIM motif. Following activation, CXCR4 is normally phosphorylated on Ser/Thr residues, which promotes receptor desensitization and internalization. We found previously that Ser/Thr phosphorylation of CXCR4 is delayed upon SDF gradient sensing, and correlates with sustained signaling to SHP2, a protein that drives hyperproliferation and invasion of breast cancer. Since SHP2 is known to be recruited to tyrosine phosphorylated ITIM motifs (immmunoreceptor tyrosine-based inhibitory consensus motifs), we investigated if CXCR4 contains an ITIM motif, and if tyrosine phosphorylation within this motif regulates SHP2 binding, signaling, and migration. Specifically, we assessed 1) if CXCR4 is tyrosine phosphorylated as measured by recombinant phosphatase assay, phospho-Ab development, and tyrosine mutagenesis (YF); 2) if tyrosine mutagenesis alters CXCR4 membrane localization, internalization, and SDF gradient sensing, by ELISA; 3) if tyrosine mutagenesis disrupts CXCR4 interaction with SHP2, by co-IP; 4) if tyrosine mutagenesis alters CXCR4 sustained signaling to SHP2, by Western; and 5) if tyrosine mutation (YF) within the novel ITIM motif in CXCR4 alters migration of metastatic breast cancer cells, by transwell motility assays. Our results demonstrate that CXCR4 phosphorylation is sensitive to recombinant tyrosine phosphatase treatment and tyrosine mutagenesis, suggesting that CXCR4 is indeed tyrosine phosphorylated within the ITIM motif. We determined that while tyrosine phosphorylation within the ITIM motif is not required for CXCR4 membrane localization, internalization, or SDF gradient sensing, it does regulate CXCR4 binding and signaling to SHP2. Investigating the role in CXCR4 mediated migration is on going. To date, our data support a working model that tyrosine phosphorylation of CXCR4 within an ITIM motif is critical for binding to SHP2 and transducing sustained signaling to SHP2. These data have implications on aggressive breast cancers with dysregulated CXCR4 and SHP2. These studies were supported by NIH grant GM-097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund.

Citation Format: Ryan D. Paulukinas, Lili T. Belcastro, Catherine C. Moore. Investigating a novel GPCR ITIM motif in regulating Shp2 binding, signaling, and migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 341. doi:10.1158/1538-7445.AM2017-341
Abstract 3414: Novel MGMT variant association in Mexican patients with astrocytoma
Authors: Gomez-Flores-Ramos Liliana , Wegman-Ostrosky Talia
Published: July 2017

Introduction: Astrocytomas are the most common and lethal brain tumors with a median survival of 15 months. Different biomarkers have arisen to classify and treat this type of tumors; one of these markers is MGMT. The methylation status of this gene is a prognostic and predictive factor. The MGMT (O-6-Methylguanine-DNA Methyltransferase) is involved in the cellular response to the effects of O6-methylguanine (O6-MeG) in DNA. Repairs alkylated guanine in DNA by transferring the alkyl group at the O-6 position to a cysteine residue in the enzyme. The dysregulation of this methylation is critical in the development of certain cancers.

Objective: To identify germinal MGMT gene variations in patients with astrocytoma.

Material and methods: 55 randomly selected Mexican patients diagnosed with astrocytoma between 2008-2014 were consented to perform the molecular analysis of the whole gene and 5´UTR and 3´UTR regions of MGMT at Ion Torrent platform. Each amplified region had a depth of minimum 1500x. Ion Reporter software was used to analyze the genetic variants and using Integrative Genomic Viewer we confirmed that each variant had a Phred higher than 25. Genotypic frequencies were compared with HapMapMex and Mexican ancestry population from the 1000 Genomes project.

Results. In lymphocytes DNA, eight genetic variants were found, and two of them were statistically significant risk factors: rs7896488 alternative allele A with a frequency of 15% in patients with astrocytoma, and only 4% of the control group (OR=5.53, IC 1.96-15.59 p = 0.0007) and rs2308326 ancestral allele C, where 93% of the patients were carriers of allele C in contrast with 73% of controls (OR=5.39, IC 2.29-12.69 p = 0.000019). These variants are non-coding single nucleotide polymorphisms, rs7896488 is a 3' UTR genetic variant, while rs2308326 is an intronic variant.

Conclusion: Our study found two new risk SNV for Astrocytoma in a Mexican population.

Citation Format: Liliana Gomez-Flores-Ramos, Talia Wegman-Ostrosky. Novel MGMT variant association in Mexican patients with astrocytoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3414. doi:10.1158/1538-7445.AM2017-3414
Abstract 3424: Role of miR-489 in mammary gland development and Her2 mediated tumorigenesis
Authors: Patel Yogin , Soni Mithil , Liu Shou , Chen Hexin
Published: July 2017

HER2 overexpression is linked with poor prognosis and outcome in breast cancer. In our previous study, we have found miR-489 was specifically down regulated by HER2 overexpression. Restoration of miR-489 in multiple breast cancer cell lines significantly inhibited cell growth in vitro and decreased tumor growth in xenograft mice. To study role of miR-489 in Her2 mediated tumorigenesis, for the first time we generated MMTV-miR-489 transgenic mice, which overexpress miR-489 specifically in mammary gland. Our qRT-PCR data has confirmed transgenic mice have significantly more miR-489 expression than FVB mice. We used western blot to further validate our model system and found significant downregulation of miR-489 targets DEK and PTPN11. To find out whether miR-489 has any role in mammary gland development, mammary gland whole mount was performed from wild type FVB mice and MMTV-miR-489 mice at different age. Mammary gland from MMTV-miR-489 mice demonstrated reduction in growth at early age. Our immunohistochemistry staining demonstrated significantly reduction in Ki-67 positive cells in MMTV-miR-489 mammary gland at early age, further confirming reduced growth in MMTV-miR-489 mice. However, we found no significant effect on weight of litters of MMTV-miR-489 female since after 8-week mammary gland was able to recover growth. To find out effect of miR-489 overexpression on Her2 mediated tumorigenesis, we generated double transgenic mice MMTV-Her2/miR-489 by crossing MMTV-miR-489 mice with MMTV-Her2 mice. We have observed significant delay in tumor onset and reduced tumor growth in MMTV-Her2/ miR-489 mice compare to MMTV-Her2 mice. We have also observed less number of metastatic site in lung by performing H and E staining of lung. Our IHC data showed reduction in PTPN11 and DEK in miR-489 overexpress mammary tumor. Surprisingly, we found significant reduction of miR-489 expression in mammary tumors of MMTV-Her2-miR-489 when compared with normal mammary gland of same mice. Overall, our results indicated miR-489 overexpression suppresses mammary gland development at early age, reduced mammary tumorigenesis and decrease lung metastasis by targeting PTPN11 and DEK.

Citation Format: Yogin Patel, Mithil Soni, Shou Liu, Hexin Chen. Role of miR-489 in mammary gland development and Her2 mediated tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3424. doi:10.1158/1538-7445.AM2017-3424
Abstract 3428: Tamoxifen differentially regulates miR-29b-1 and miR-29a depending on endocrine sensitivity in breast cancer
Authors: Muluhngwi Penn , Klinge Carolyn M.
Published: July 2017

Acquired endocrine-resistance occurs in ~ 40% of estrogen receptor α (ERα)+ breast cancer patients after tamoxifen (TAM) or aromatase inhibitor therapy. MicroRNAs (miRs) are dysregulated in breast cancer, but their contribution to endocrine-resistance is not yet completely understood. Microarray analysis of miRNAs in TAM-sensitive MCF-7 versus TAM-resistant LY2 breast cancer cells (derived from MCF-7 cells) identified differential regulation of miR-29b-1 and miR-29a. miR-29a and miR-29b-1, which are co-transcribed as a single primary-miR, were repressed by TAM in MCF-7 and stimulated by TAM in LY2 cells. We confirmed these observations by real time qPCR in MCF-7 and LY2 cells and observed that 4-OH-TAM (4-OHT) also downregulated miR-29b-1/a in LCC2 and LCC9 endocrine-resistant cells. These changes were at the primary transcriptional level. The role of these two miRs in endocrine-resistant breast cancer is unknown. We tested the hypothesis that the TAM-stimulated increase in miR-29b-1/a promotes endocrine-resistance by downregulating targets that contribute to TAM’s antiproliferative/pro-apoptotic activity. Our goals were to: 1) Determine if ERα was responsible for TAM regulation of miR-29b-1/a expression; 2) Determine the functional role(s) of miR-29b-1/a; 3) Identify specific miR-29b-1/a target genes that are differentially regulated by 4-OHT in MCF-7 versus LCC9 and LY2 cells. Knockdown of ERα blocked 4-OHT inhibition of miR-29b-1/a in MCF-7 and 4-OHT simulation of miR-29-b-1/a transcription in LCC9 and LY2 cells. Transient over-expression of miR-29b-1/a inhibited proliferation of MCF-7, LCC9, and LY2 cells and decreased migration and colony formation of LY2 cells. Repression of miR-29b-1/a has no significant effect on MCF-7, LCC9, or LY2 cell proliferation. Repression of miR-29-b-1/a did not sensitize the TAM-resistant cells to growth inhibition by 4-OHT. We observed that the bona fide miR-29 target gene, DICER1, was inhibited by 4-OHT in LY2 cells and anti-miR29b-1 or anti-miR-29a decreased the suppression of DICER1 by 4-OHT. These data suggest that 4-OHT-ERα increases miR-29b-1/a which target and downregulate DICER 1 in endocrine resistant breast cancer cells. Future studies will identify other targets of miR-29b-1/a to better understand the pathways leading to acquired endocrine resistance.

Citation Format: Penn Muluhngwi, Carolyn M. Klinge. Tamoxifen differentially regulates miR-29b-1 and miR-29a depending on endocrine sensitivity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3428. doi:10.1158/1538-7445.AM2017-3428
Abstract 3429: Oncogenic function of miR-23b-3p in hepatocellular carcinoma
Authors: Tanabe Hiroshi , Hayashi Masamichi , Sugimoto Hiroyuki , Kurimoto Keisuke , Hirabayashi Sho , Kanda Mitsuro , Takami Hideki , Niwa Yokiko , Iwata Naoki , Kobayashi Daisuke , Tanaka Chie , Yamada Suguru , Nakayama Goro , Koike Masahiko , Fujii Tsutomu , Fujiwara Michitaka , Kodera Yasuhiro
Published: July 2017

Background: miR-23b has been identified as various cancer-related biomarkers. Interestingly, it works as an oncogenic miRNA in lymphoma, renal cancer and glioma, while it works as a tumor suppressor miRNA in pancreatic cancer, bladder cancer and prostate cancer (Donadeli M et al, Cancer Lett, 2014). We have previously reported miR-23b-3p as a oncomiR in NSCLCs (Begum S, Hayashi M et al, Sci Rep, 2015). To find the correlation to other carcinogenesis, we focused on miR-23b function in hepatocellular carcinoma (HCC).

Methods: miR-23b-3p expression was examined in 9 HCC cell lines (SK-Hep1, HuH2, HLE, PLC/PRF/5, HuH1, HuH7, HLF, Hep3B, HepG2). The downstream of miR-23b-3p overexpression was examined by Cancer Pathway Finder (Qiagen). Results were applied to 125 clinical HCC samples (2002-2011 surgically resected).

Results: Transfection experiments were performed for HepG2 (miR-23b-3p highly expressed) by siRNA and for SK-Hep1 (lowly expressed) by miRNA mimic. Cancer cell proliferation was activated by miR-23b-3p overexpression, and diminished by its inhibition. In order to find the associated genes, miR-23b-3p overexpressed SK-Hep1 cells were compared with parental SK-Hep1 cells by global gene expression analysis. ANGP1, ERCC5 and G6PD genes were upregulated, while KDR, WEE1, OCLN genes were downregulated. We also detected additional two genes (AUH and MICU3) by TargetScan Release 5.2. Clinical HCC samples were divided into miR-23b-3p upregulated 48 cases (38%) and downregulated 77 cases (62%). Upregulated cases were correlated with aged patients (P=0.015), capsule invasion positive (P=0.039) and serosal invasion positive (P=0.049). Also, they showed significantly poor recurrence free survival (HR=1.64, P=0.037, 95%CI:1.03-2.59) and overall survival (HR=3.10, P=0.001, 95%:1.57-6.29) in multivariable analysis.

Conclusion: miR-23b-3p increased the HCC cell proliferation, and indicated the invasive type of HCCs. It functions as a oncogenic biomarker in HCCs and might be a therapeutic target.

Citation Format: Hiroshi Tanabe, Masamichi Hayashi, Hiroyuki Sugimoto, Keisuke Kurimoto, Sho Hirabayashi, Mitsuro Kanda, Hideki Takami, Yokiko Niwa, Naoki Iwata, Daisuke Kobayashi, Chie Tanaka, Suguru Yamada, Goro Nakayama, Masahiko Koike, Tsutomu Fujii, Michitaka Fujiwara, Yasuhiro Kodera. Oncogenic function of miR-23b-3p in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3429. doi:10.1158/1538-7445.AM2017-3429
Abstract 3446: Melatonin suppresses hepatocellular carcinoma progression via lncRNA-CPS1-IT-mediated HIF-1α inactivation
Authors: Wang Tong Hong , Wu Chi Hao , Yeh Chau Ting , Liang Kung Hao , Hsueh Chuen , Chen Chi Yuan
Published: July 2017

Background: Melatonin, the main pineal hormone that relays light/dark cycle information to the circadian system, was recently reported to have intrinsic antitumor activity in various cancers. However, the detailed regulatory mechanisms associated with antitumor activity of melatonin are not well understood yet. Moreover, limited studies have addressed the role of melatonin in hepatocellular carcinoma (HCC), the major life-threatening malignancy for both sexes in Taiwan. Hence, in this study, we investigated the antitumor activity of melatonin on HCC and explored its regulatory mechanism.

Methods: Human hepatoma cell lines HepG2 and Huh7 were treated with melatonin followed by functional assays to dissect antitumor effect of melatonin in HCC. The whole transcriptome sequence analysis was performed to explore the long non-coding RNAs (lncRNAs) involved in the anti-HCC activity of melatonin. Further, quantitative RT-PCR, western blot, immunohistochemistry assays, and in vivo animal model experiments were performed to elucidate the regulatory mechanism of lncRNAs in melatonin-mediated HCC suppression.

Results: Melatonin significantly inhibited cell proliferation, migration, and invasion capacity of HCC cell lines, while the expression of transcription factor FOXA2 was significantly induced in cells treated with melatonin. The increase in expression of FOXA2 resulted in upregulation of lncRNA-CPS1-IT1 expression, which in turn reduced binding and activation of Hsp90 with HIF-1α. The inactivation of HIF-1α resulted in the suppression of epithelial-mesenchymal transition (EMT) progression and HCC metastasis. Furthermore, the results of the in vivo animal model experiments also validated the tumor-suppressor role of melatonin via a reduction in tumor growth.

Conclusions: Taken together, our findings suggested that melatonin inhibited HCC progression by modulating lncRNA-CPS1-IT1-mediated EMT suppression and further supported melatonin as a promising therapeutic agent for the treatment of HCC.

Note: This abstract was not presented at the meeting.

Citation Format: Tong Hong Wang, Chi Hao Wu, Chau Ting Yeh, Kung Hao Liang, Chuen Hsueh, Chi Yuan Chen. Melatonin suppresses hepatocellular carcinoma progression via lncRNA-CPS1-IT-mediated HIF-1α inactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3446. doi:10.1158/1538-7445.AM2017-3446
Abstract 3452: The cohesin subunit STAG1 is a hardwired genetic dependency of STAG2 mutant cancer cells
Authors: Lelij Petra Van Der , Lieb Simone , Jude Julian , Wutz Gordana , Pereira Catarina , Falkenberg Katrina , Ban Jozef , Kovar Heinrich , Waldman Todd , Real Francisco , Pearson Mark , Kraut Norbert , Peters Jan-Michael , Zuber Johannes , Petronczki Mark P.
Published: July 2017

Recent genome analyses have identified recurrent mutations in subunits of the cohesin complex in human cancer. Cohesin is a chromosomal factor that is essential for sister chromatid cohesion and cell division and that contributes to gene regulation and DNA repair. Deleterious mutations in the cohesin subunit STAG2 have been detected in about 20% of bladder cancer, 15% of Ewing sarcoma and 6% of AML/MDS patients. The mechanistic involvement of cohesin mutations in the pathogenesis of human malignancies is currently under active investigation. We hypothesized that the loss of STAG2 could alter the properties and functionalities of the cohesin complex leading to unique vulnerabilities of STAG2 mutant cells. Using CRISPR/Cas9 and RNAi in isogenic solid cancer and leukemic models we identified STAG1, a STAG2 paralog, as a strong and clean genetic vulnerability of STAG2 mutant cells. Mechanistically, STAG1 loss abrogates sister chromatid cohesion specifically in STAG2 mutant but not wild-type cells leading to mitotic catastrophe, defective cell division and apoptosis. STAG1 inactivation inhibits the proliferation of disease relevant STAG2 mutant but not wild-type bladder cancer and Ewing sarcoma cell lines. Restoration of STAG2 expression in a mutant bladder cancer model alleviates the dependence on STAG1. Our results demonstrate that the cohesin subunits STAG1 and STAG2 act redundantly to support sister chromatid cohesion and cell viability in human cells. We have identified STAG1 as a hardwired, context independent vulnerability of STAG2 mutant cancers. Specific synthetic lethalities elicited by recurrent cohesin mutations in human tumors hold the promise for the development of selective therapeutics.

Citation Format: Petra Van Der Lelij, Simone Lieb, Julian Jude, Gordana Wutz, Catarina Pereira, Katrina Falkenberg, Jozef Ban, Heinrich Kovar, Todd Waldman, Francisco Real, Mark Pearson, Norbert Kraut, Jan-Michael Peters, Johannes Zuber, Mark P. Petronczki. The cohesin subunit STAG1 is a hardwired genetic dependency of STAG2 mutant cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3452. doi:10.1158/1538-7445.AM2017-3452
Abstract 3455: Cystatin C decreases proliferation of melanoma cells by affecting mitosis length
Authors: Wallin Hanna , Alm Kersti , Abrahamson Magnus
Published: July 2017

Cystatin C is a small secreted protein, produced by most cells in the body and found in significant quantities in all body fluids. It is the major inhibitor of extracellular cysteine protease activity in mammals because of its rapid binding to virtually all known cysteine proteases of families C1 (cysteine cathepsins) and C13 (mammalian legumain). Cystatin C has also been shown to inhibit viral replication within cells and display protective effects against cancer cell invasion. The protein has recently been shown to get internalized into cancer cells, providing an explanation to possible intracellular effects of the inhibitor.

The purpose of the present study was to elucidate possible direct effects of cystatin C on cancer cell growth, by use of holometric imaging to analyze proliferation rate as well as to monitor the mitosis phase in real-time. Human A375 melanoma cells were cultured with and without addition of physiological quantities of cystatin C added to the culture medium (1 micromolar final concentration). The cultures were monitored for up to 3 days in a standard incubator using a HoloMonitor M4 instrument. Settings were optimized to allow accurate counting of cells throughout the experiment by analysis of images captured, revealing an inhibitory effect on overall cell proliferation rate by cystatin C addition to the cultures. To analyze the migration and cell division of individual cells, images were captured at short intervals allowing tracking and monitoring of cell morphology with respect to parameters such as thickness/density and area. Parameters could be set to define start of the mitosis phase indicated by a distinct rounding-up of cells and followed until a natural stop at cytokinesis, whereafter the daughter cells could be continuously tracked. These analyses revealed qualitative differences in the normal migration behavior of the A375 cells under study as a result of cystatin C addition. Moreover, the mitosis phase was considerably prolonged in cells grown in cystatin C containing medium, indicating that the proliferation-inhibiting effect of the protease inhibitor may be through its direct action on proteolytic processes in the actual mitosis phase. This finding justifies in-depth studies on additional cancer cell types to reveal how general the anti-proliferative effect of cystatin C is and how it can be explored to possibly control cancer cell growth and spread. The use of real-time holometric imaging of growing cells should be a most useful tool in such studies.

Citation Format: Hanna Wallin, Kersti Alm, Magnus Abrahamson. Cystatin C decreases proliferation of melanoma cells by affecting mitosis length [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3455. doi:10.1158/1538-7445.AM2017-3455
Abstract 3467: A DAXX-KIFC3 fusion potentiates alternative lengthening of telomeres in osteosarcoma
Authors: Soper Sarah F. Clatterbuck , Showman Soyeon A. , Driest Kathryn E. , Waterfall Joshua J. , Walker Robert L. , Pineda Marbin A. , Zhu Yuelin J. , Wang Yonghong , Ester Corbin D. , Bilke Sven , Meltzer Paul S.
Published: July 2017

Proliferating cells must enact a program of telomere lengthening to counteract the chromosome end replication problem. In most types of cancer cells, telomeres are maintained through the action of the ribonucleoprotein telomerase, but some cancer cells, particularly those of mesenchymal origin, utilize an alternative method of telomere repair and lengthening termed the alternative lengthening of telomeres (ALT) pathway. Since telomere maintenance is essential for tumor cell immortality, better understanding of the ALT mechanism could potentially reveal drug targets that could be used to develop novel therapies for tumors that use ALT. It has been previously observed that ALT tumors frequently carry mutations in ATRX, which partners with the protein DAXX in a chromatin remodeling complex, but how these mutations facilitate the ALT pathway is not well understood. Work in our lab identified an ALT-positive osteosarcoma cell line, identified here as OS1, in which DAXX has undergone a fusion event with the non-canonical kinesin KIFC3. We find that knockdown of the DAXX-KIFC3 fusion neither impairs ALT nor cell proliferation, suggesting that the fusion represents a loss of function. Furthermore, inducible restoration of wild-type DAXX, reversibly abrogates ALT function in this cell line. One of the hallmarks of ALT is localization of telomeres and DNA recombination machinery to nuclear PML bodies, resulting in formation of ALT- associated PML Bodies, or APBs. Thus it may be considered that changes in PML body composition represent a key aspect of the ALT mechanism. We observe that in OS1 both DAXX and ATRX fail to localize to PML bodies. This finding is consistent with the fact that the DAXX-KIFC3 fusion results in loss of a C-terminal SUMO interaction motif that normally mediates PML body interaction. Leveraging our inducible system, using biochemical and imaging approaches, we are working to define the role of DAXX in maintaining PML body composition.

Citation Format: Sarah F. Clatterbuck Soper, Soyeon A. Showman, Kathryn E. Driest, Joshua J. Waterfall, Robert L. Walker, Marbin A. Pineda, Yuelin J. Zhu, Yonghong Wang, Corbin D. Ester, Sven Bilke, Paul S. Meltzer. A DAXX-KIFC3 fusion potentiates alternative lengthening of telomeres in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3467. doi:10.1158/1538-7445.AM2017-3467
Abstract 3489: Noncoding RNA distribution in clear cell renal cell cancer: small RNA-seq data
Authors: Koduru Srinivas V. , Ravnic Dino J.
Published: July 2017

Small noncoding RNAs (sncRNAs) play pivotal roles in biological processes and may prove to be a tool in cancer diagnosis, prognosis, and treatment. Noncoding RNAs such as microRNAs (miRNAs), piwi-interacting-RNAs (piRNAs), long noncoding RNA (lncRNAs) and small nuclear/nucleolar RNAs (sn/snoRNAs) have recently been investigated to identify their role in cancer. Approximately 62,700 new cases of kidney cancer are expected each year in the United States leading to 14,240 deaths, with rates rising over the past 20 years. Nearly 90% of all kidney cancers will be renal cell carcinoma, of which 70% will be a clear cell renal cell carcinoma (ccRCC) subtype. When detected early most cases can be treated effectively, however there is a lack of early detection tests. We sought to identify molecular markers for early detection and diagnosis. We downloaded publically available (NIH bioproject PRJNA162397) ccRCC small RNA sequence raw data for noncoding RNA analysis. 22 ccRCC and 11 non-tumor renal cortex patient samples were evaluated. Data was analyzed by PartekFlow software. sRNA-seq data was annotated to miRNAs with 69 miRNAs aberrantly expressed in ccRCC with 37 being upregulated (top five: miR-122, miR-210, miR155, miR-224 and miR-21) and 32 being downregulated (top five: miR-200c, miR-502, miR-20b, miR10a and miR-204). Utilizing MetaCore software for pathway analysis the majority of expressed miRNAs were involved in metabolism related signaling pathways. Examining data for piRNAs revealed 22 which were significantly expressed (8 upregulated and 14 downregulated). Investigation of lncRNAs revealed 15 to be downregulated and 27 upregulated. We also uncovered 2 mitochondrial rRNAs, 10 miscRNAs, 1 snRNA, 7 snoRNAs, and 5 rRNAs in ccRCC samples. Our comprehensive analysis of publically available small RNA-seq data identified numerous sncRNAs associated with ccRCC. Pending further validation, they may prove useful as early detection biomarkers in this common cancer.

Citation Format: Srinivas V. Koduru, Dino J. Ravnic. Noncoding RNA distribution in clear cell renal cell cancer: small RNA-seq data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3489. doi:10.1158/1538-7445.AM2017-3489
Abstract 3490: Interrogation of small RNA-seq data for small noncoding RNA in human colon cancer
Authors: Koduru Srinivas V. , Nyinawabera Angelique , Ravnic Dino J. , Tiwari Amit K.
Published: July 2017

Genomic analysis of the human transcriptome has been made possible only in last decade by next generation sequencing (NGS). Recent advancements in NGS has further allowed us to look into small non-coding RNAs (sncRNAs) such as microRNAs (miRNAs), Piwi-interacting-RNAs (piRNAs), long non-coding RNAs (lncRNAs) & small nuclear/nucleolar RNAs (sn/snoRNAs). Recently, the roles of sncRNAs in biological processes have been implicated in biomarker development for diagnosis, prognosis &therapy. In the present study, 50 colon cancer patient’s small RNA sequencing raw data was downloaded from NIH bioproject (PRJNA266667; 7 TNM stage II & 43 TNM stage III) which contained 27 female & 23 male samples. 24 samples had metachronous metastasis (MM) & 26 non-metachronous metastasis (NMM). The small RNA-seq data was analyzed using PartekFlow. In depth analysis showed aberrant expression of 48 miRNAs (all upregulated) in TNM-III vs TNM-II specimens & 20 miRNAs (17 upregulated & 3 downregulated) in MM vs NMM. Further investigation of dysregulated miRNA through pathway analysis confirmed that the majority of the miRNAs were involved in cancer signaling pathways. Analysis of piRNA found unusual expression of 60 piRNAs (57 upregulated & 3 downregulated) in TNM-III vs TNM-II & 31 piRNAs (28 upregulated & 3 downregulated) in MM vs NMM. Further analysis of long non-coding RNAs, we found 77 lncRNAs were significantly expressed in TNM-III vs TNM-II &18 lncRNAs in MM vs NMM. We also, investigated small nuclear/nucleolar RNAs (sn/snoRNAs), miscRNAs & mtRNAs, we identified 37 snRNAs, 105 snoRNAs, 28 miscRNAs & 5 mtRNAs in TNM-III vs TNM-II whereas 2 snRNAs, 11 snoRNAs, 57 miscRNAs & 8 mtRNAs in MM vs NMM were identified. In summary, our comprehensive analysis on publicly available small RNA-seq data identified multiple small non-coding RNAs that need to be further explored for their use in the prognosis, diagnosis & therapy of colon cancer.

Citation Format: Srinivas V. Koduru, Angelique Nyinawabera, Dino J. Ravnic, Amit K. Tiwari. Interrogation of small RNA-seq data for small noncoding RNA in human colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3490. doi:10.1158/1538-7445.AM2017-3490
Abstract 3496: Long non-coding RNAs in colorectal cancer
Authors: Opattova Alena , Ferreira Fábio Miguel , Horak Jozef , Vodenkova Sona , Vodicka Pavel
Published: July 2017

Colorectal cancer (CRC) is the third most common type of cancer worldwide and the second most common cause of cancer related deaths in Europe. Since Countries of Central Europe (Czech Republic, Slovakia, Hungary) have one of the highest rates both for incidence and mortality of CRC, CRC became a serious health, social and economic problem. CRC pathogenesis involves multiple genetic events. On behalf of a strong experimental work, there is still lack of predictive, prognostic and treatment-response related biomarkers. Long non-coding RNAs (lncRNA) are non-protein coding transcripts longer than 200 nucleotides. Deregulation of lncRNAs is often seen in a variety of human diseases, including cancer. Taken together, this makes lncRNAs the potential regulators of cancer progression, therapeutic targets or markers of treatment efficacy.

The main aim of our present study was to define expression patterns of lncRNAs in colorectal tumor tissue and define their role in cancer cell proliferation, survival and DNA damage response (DDR).

In the first part of our study, we analyzed the expression profile of 83 lncRNA known to be associated with cancer progression in cohort of CRC tumor tissues and adjacent mucosa. We found 12 upregulated lncRNAs (p<0.05) and 10 downregulated lncRNAs (p<0.05). Our results have been replicated on larger set of patients (100 CRC patients) and those lncRNAs associated with cell proliferation, survival and DNA damage response are functionally tested on colorectal cell lines (HCT116, HCT116 p53-/-) in vitro.

Definition of role of lncRNA as a tumor suppressors or oncogenes in CRC etiology may lead to better understanding of regulation of CRC carcinogenesis. Furthermore, understanding the role of lncRNA in DDR may contribute to better treatment response of CRC patients and to define markers of individual treatment response.

Acknowledgement: GACR 15-14789S, AZV 15-27580A, GAUK800216, AZV 15-26535A, SUSCEPTCOST LD14050

Citation Format: Alena Opattova, Fábio Miguel Ferreira, Jozef Horak, Sona Vodenkova, Pavel Vodicka. Long non-coding RNAs in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3496. doi:10.1158/1538-7445.AM2017-3496
Abstract 3503: Transcriptome analysis reveals differentially expressed lncRNAs between oral squamous cell carcinoma and healthy oral mucosa
Authors: Feng Lu , Houck John R. , Lohavanichbutr Pawadee , Chen Chu
Published: July 2017

Objective: Oral cavity and oropharyngeal squamous cell carcinoma (OSCC) is a major cancer type in the head and neck region. To better understand the roles long non-coding RNA (lncRNA) play in OSCC carcinogenesis, we compared the genome-wide gene expressions of lncRNA in OSCC and in the oral mucosa from healthy individuals.

Materials and methods: We compared the expression levels of 3,054 probe sets for lncRNAs between 167 OSCCs and 45 healthy controls using an Affymetrix HG U133 plus 2.0 array dataset. We validated the top differentially expressed lncRNAs in three independent datasets in the Gene Expression Omnibus (GEO) repository: GSE42743, GSE9844, and GSE6791. We further tested the differential expression of the three lncRNAs showing the highest fold change by quantitative RT-PCR in a subset of 20 OSCCs and 10 control samples.

Results: We found 658 lncRNA transcripts (790 probe sets) to be significantly differentially expressed between OSCC and healthy oral mucosa using a criteria of FDR<0.01, with 36 of them (39 probe sets) showing more than a 2-fold change. Among the 36, 14 (15 probe sets) lncRNAs were validated in all three independent datasets using the criteria FDR<0.01: LOC441178, C5orf66-AS1, HCG22, FLG-AS1, CCL14/CCL15-CCL14, LOC100506990, TRIP10, PCBP1-AS1, LINC01315, LINC00478, COX10-AS1/LOC100506974, MLLT4-AS1, MIR31HG, and DUXAP10/LINC01296. The differential expressions of LOC441178, HCG22 and C5orf66-AS1 were verified by qRT-PCR.

Conclusion: We identified a number of differentially expressed lncRNAs using transcriptomic data between OSCCs and healthy oral mucosa. Further investigations into their potential role in OSCC carcinogenesis or to serve as OSCC biomarkers and/or therapeutic targets are warranted.

Citation Format: Lu Feng, John R. Houck, Pawadee Lohavanichbutr, Chu Chen. Transcriptome analysis reveals differentially expressed lncRNAs between oral squamous cell carcinoma and healthy oral mucosa [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3503. doi:10.1158/1538-7445.AM2017-3503
Abstract 3509: EWS/FLI regulates transcriptional activation via length-dependent GGAA microsatellites
Authors: Johnson Kirsten M. , Taslim Cenny , Lessnick Stephen L.
Published: July 2017

Objective: The purpose of this study is to investigate how EWS/FLI transcriptionally activates gene targets via polymorphic GGAA microsatellites. Ewing Sarcoma is a pediatric bone malignancy initiated by a t(11;22) chromosomal translocation that produces the EWS/FLI oncoprotein. EWS/FLI transcriptionally activates and represses its target genes to mediate oncogenic reprogramming. Expression of its up-regulated targets correlates with EWS/FLI binding to associated GGAA microsatellites, which show length polymorphisms. These microsatellite polymorphisms may critically affect EWS/FLI-responsiveness of key gene targets. For example, NR0B1 is necessary for EWS/FLI mediated oncogenic transformation, and we found a “sweet-spot” of 20-25 repeat length as optimal for EWS/FLI mediated transcriptional activity at NR0B1 through clinical observation and in vitro studies. The mechanism underlying this optimal length is unknown.

Methods: We explored the stoichiometry and binding affinity of EWS/FLI for different repeat lengths through biochemical studies, including fluorescence polarization, and immunoprecipitation assays, combined with bioinformatics analysis. Additionally, use of EWS/FLI mutant constructs has been critical for elucidating particular binding behavior of EWS/FLI at different microsatellite repeat lengths.

Results: Fluorescence anisotropy studies demonstrate that FLI binding affinity is independent of GGAA microsatellite length. In contrast, the stoichiometry of protein to DNA binding increases in specific incremental patterns with increasing microsatellite repeats. EWS/FLI mutants inform on binding, suggesting a complex relationship between microsatellite length and transcriptional activity.

Conclusion: Overall our data suggests a model in which the DNA binding domain of multiple FLI monomers function as independent binding units to facilitate transcriptional activity in a length-dependent manner. The EWS portion appears to be critical for in vivo binding of genomic DNA globally. We propose that GGAA microsatellites are necessary and sufficient for EWS/FLI-mediated oncogenic transformation and that repeat length affects optimal DNA binding stoichiometry and transcriptional activity.

Citation Format: Kirsten M. Johnson, Cenny Taslim, Stephen L. Lessnick. EWS/FLI regulates transcriptional activation via length-dependent GGAA microsatellites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3509. doi:10.1158/1538-7445.AM2017-3509
Abstract 3528: PTCH53 as a potential secondary modifier in Li-Fraumeni syndrome
Authors: Pan Anna J. , Brew Benjamin , Erdman Lauren , Goldenberg Anna , Malkin David
Published: July 2017

Germline mutations in the TP53 gene have been established as the underlying genetic event in Li-Fraumeni syndrome (LFS), predisposing affected families to a wide spectrum of early onset cancers. Despite rapid progress in elucidating the role of wild-type p53 in maintenance of genome stability, and mutant p53 in cellular transformation, much of the molecular basis underlying LFS remains unclear. Lack of predictability in age of onset, type(s) of cancers, and likelihood of subsequent malignancies prompts further research to examine secondary modifiers on the underlying TP53 genotype. Patched-domain containing 4 (a.k.a PTCHD4 or PTCH53) is a repressor of canonical Hedgehog signaling that is also described to be transcriptionally activated by p53. Furthermore, our group has identified significant hypomethylation at the PTCH53 locus in a mutant TP53 LFS cohort, compared to TP53 wildtype carriers. These results suggest a potential role of PTCH53 in modifying LFS phenotype. The study examines the expression profile of PTCH53 in the presence of different germline TP53 mutations using primary LFS patient-derived fibroblasts, and aims to elucidate the functional role of PTCH53. PTCH53 transcriptional expression was assessed using digital droplet PCR in 10 LFS non-transformed fibroblast cell lines representing a spectrum of heterozygous germline TP53 mutations. Our data demonstrate that PTCH53 expression indeed depends on TP53 genotype where different TP53 mutations correspond with varied PTCH53 mRNA levels. Nonetheless, mutant TP53 fibroblasts have overall lower PTCH53 transcripts compared to wild-type TP53 fibroblasts. Fold-change increase in PTCH53 expression was detected upon doxorubicin and γ-irradiation induced activation of the p53 protein. These results suggest that induction of p53 via DNA damage activates a signaling cascade involving PTCH53 up-regulation. Further experiments will examine the functional consequences to PTCH53 de-regulation via shRNA knockdown and CRISPR knockout models, and will examine possible modifying effects of PTCH53 on the functional phenotype (cell viability, colony formation and migration potentials) of cell lines harboring different p53 mutants. Elucidating the functional profile of PTCH53 in the context of Li-Fraumeni syndrome will contribute to understanding the complex molecular basis underlying this cancer predisposition syndrome.

Citation Format: Anna J. Pan, Benjamin Brew, Lauren Erdman, Anna Goldenberg, David Malkin. PTCH53 as a potential secondary modifier in Li-Fraumeni syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3528. doi:10.1158/1538-7445.AM2017-3528