bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1036: Cellular phenotypes of a novel series of 3-phosphoglycerate dehydrogenase inhibitors
Authors: Mainolfi Nello , Friedman Adam , Moyer Mikel P. , Suri Vipin , Manfredi Mark
Published: July 2016

PHGDH (3-phosphoglycerate dehydrogenase) is the first enzyme branching from glycolysis into the serine synthetic pathway and it oxidizes 3-phosphoglycerate into phospho-hydroxypyruvate using nicotinamide adenine dinucleotide (NAD) as cofactor.[1] Increases in PHGDH expression at both mRNA and protein levels have been observed in nearly 70% of estrogen receptor-negative breast cancers; in addition a fraction of malignant breast and melanoma cells are dependent on elevated expression of 3-phosphoglycerate dehydrogenase (PHGDH).[2] Furthermore, serine starvation has been shown to have a dramatic effect on tumor growth during in vivo mouse xenograft experiments.[3] PHGDH has been a target of interest in the pharma/biotech industry for several years since the initial reports in early 2012 of its relevance in cancer2 where PHGDH amplified and overexpressing cancer cell lines have been shown to possess unique sensitivity to PHGDH knockdown that cannot be rescued by nutritional serine. The mechanisms underlying these studies have been subject to intense investigation but remain unclear. We have been able to successfully identify first in class small molecule inhibitors of this target with nanomolar cellular potency, high degree of selectivity and oral bioavailability. In several cancer cell lines, these compounds inhibited glucose derived serine flux with nanomolar median inhibitory concentrations without significantly affecting glucose derived lactate. These compounds also inhibited glucose derived serine in animal studies and have the potential to be highly useful tools for understanding the role of PHGDH in tumor progression. The data presented here will provide unexpected insights on the role of PHGDH in serine biosynthesis and the dependency of cancer cells on PHGDH catalytic function.

References

[1] Snell, K. Enzymes of serine metabolism in normal, developing and neoplastic rat tissues. Advances in enzyme regulation. 1984; 22:325-400.

[2] Cantor, J.R.; Sabatini, D. M. Cancer Cell Metabolism: One Hallmark, Many Faces, Cancer Discovery. 2012; 2(10):881-898.

[3] Maddocks et al., Serine starvation induces stress and p53-dependent metabolic remodeling in cancer cells Nature, 2013, 493(7433):542-6

Citation Format: Nello Mainolfi, Adam Friedman, Mikel P. Moyer, Vipin Suri, Mark Manfredi. Cellular phenotypes of a novel series of 3-phosphoglycerate dehydrogenase inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1036.
Abstract 1045: Targeting NAMPT in Ewing's sarcoma cells
Authors: Mutz Cornelia N. , Schwentner Raphaela , Bouchard Eric , Mejia Edgard M. , Katschnig Anna M. , Kauer Maximilian O. , Aryee Dave N.T. , Garten Antje , Banerji Versha , Kovar Heinrich
Published: July 2016

Ewing Sarcoma (ES) is the second most common bone cancer in children and adolescents with a high metastatic potential. Tumor development is driven by the specific t(11;22)(q24;q12) chromosomal translocation resulting in the generation of the chimeric transcription factor EWS-FLI1.

NAD is a key metabolite of energy metabolism being involved in cellular redox reactions, DNA repair, and in the maintenance of genomic stability serving as a donor of ADP-ribose. This study describes targeting NAMPT (nicotinamide phosphoribosyltransferase), the rate-limiting enzyme in salvage generation of NAD, by FK866 in ES cells. Using FK866 has been proposed as a treatment option for various inflammatory diseases as well as cancer, rendering ES cells with high NAMPT expression especially susceptible to FK866-induced cytotoxicity.

Here we report that NAMPT inhibition in ES cells leads to exhaustive NAD depletion, followed by a delayed reduction of ATP levels and concomitantly to apoptosis-mediated cell death. These effects can be reversed by nicotinic acid, a substrate for the NAD salvage generation. However, the use of a doxycycline-inducible shRNA against EWS-FLI1 revealed that the cytotoxic activity of NAMPT inhibition is significantly lowered in the absence of EWS-FLI1. EWS-FLI1-low ES cells have higher viability and lower rates of apoptosis throughout inhibitor treatment compared to cells with high EWS-FLI1 expression. Additionally, changes in mitochondrial respiration and glycolytic rate can be observed when comparing untreated versus EWS-FLI1 knockdown ES cells after NAMPT inhibition. Interestingly, loss of EWS-FLI1 leads to elevated NAD levels and results in alteration of RNA expression of some enzymes involved in the NAD synthesis pathway. These results might explain the high susceptibility of Ewing Sarcoma cells to FK866 treatment.

Taken together, our data reveal evidence of an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of ES cells and suggests NAMPT inhibition as a potential new treatment approach for Ewing Sarcoma in combination with standard therapies.

Supported by the Austrian Science fund, grant I1225-B19; and the Research Manitoba and CancerCare Manitoba Foundation.

Citation Format: Cornelia N. Mutz, Raphaela Schwentner, Eric Bouchard, Edgard M. Mejia, Anna M. Katschnig, Maximilian O. Kauer, Dave N.T. Aryee, Antje Garten, Versha Banerji, Heinrich Kovar. Targeting NAMPT in Ewing's sarcoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1045.
Abstract 1049: Pharmacological characterization of a novel potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with robust in vivo efficacy and increased therapeutic index with niacin supplementation
Authors: Quanz Maria , Merz Claudia , Bernthaler Andreas , Kaulfuss Stefan , Richter Anja , Bauser Marcus , Haegebarth Andrea
Published: July 2016

Nicotinamide adenine dinucleotide (NAD+) is an important metabolite and cofactor for a number of cellular processes including genomic stability and epigenetic regulation. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate limiting enzyme in NAD+ salvaging from nicotinamide. Many tumor cells have an increased need for NAD+ and are therefore highly sensitive to NAMPT inhibition. We have developed a selective and potent small molecule inhibitor, Cmpd1, which inhibits the NAMPT enzyme with sub-nanomolar potency. In cells, Cmpd1 treatment leads to an almost complete reduction of both NAD- and ATP-levels, followed by the induction of cell death. In order to identify sensitive cancer subtypes and associated biomarkers, we analyzed cell sensitivity to NAMPT inhibition in a panel of 240 cell lines. Small cell lung cancer and hematological malignancies were particularly sensitive to NAMPT inhibition. Overall, expression levels of NAMPT and NAD+ consuming enzymes correlated well with sensitivity to NAMPT inhibition.

In order to investigate the effect of niacin on the antiproliferative effect of NAMPT inhibition across the cell panel, we further analyzed the cell sensitivity in the presence of niacin. Cells with a functional Preiss-Handler pathway can generate NAD+ from niacin, independent of NAMPT. We found that the antiproliferative effect of Cmpd1 was neutralized by niacin in 60% of the analyzed cells. The mRNA levels of nicotinate phosphoribosyltransferase 1 (NAPRT1), a central enzyme of the Preiss-Haendler pathway, predicted well the niacin rescue status of the cells. In vivo, niacin supplementation led to an at least 10-fold increase in the maximum tolerated dose of Cmpd1 in mice. Antitumor efficacy of Cmpd1 was abolished by niacin supplementation in xenografts derived from NAPRT1 expressing cells, but not in NAPRT1-deficient models. These data demonstrate that the therapeutic index of NAMPT inhibitors may be increased in NAPRT1-deficient tumors by niacin supplementation.

Citation Format: Maria Quanz, Claudia Merz, Andreas Bernthaler, Stefan Kaulfuss, Anja Richter, Marcus Bauser, Andrea Haegebarth. Pharmacological characterization of a novel potent nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with robust in vivo efficacy and increased therapeutic index with niacin supplementation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1049.
Abstract 1065: Reduced expression of microRNA-200 family associated with aggressiveness and poorer patient outcome of clear cell renal cell carcinomas
Authors: Gotoh Masahiro , Arai Eri , Matsuda Akio , Fujimoto Hiroyuki , Matsumoto Kenji , Kanai Yae
Published: July 2016

[Introduction] The aim of this study was to clarify the significance of alterations of microRNA (miRNA) expression during renal carcinogenesis.

[Materials and methods] Expression profiles of miRNA were examined using the SurePrint G3 miRNA Expression microarray in paired samples of cancerous tissue (T) and non-cancerous renal cortex tissue (N) from 95 patients with clear cell renal cell carcinomas (RCCs). The expression levels of miRNA and mRNA were quantified by reverse transcription PCR (RT-PCR) using TaqMan system. The MetaCore pathway analysis by GeneGo was performed using miRNAs of which expression levels were significantly altered during renal carcinogenesis.

[Results] The expression levels of 39 and 169 miRNAs were increased (T/N> = 2) and reduced (T/N<0.5) in T samples relative to the corresponding N samples, respectively. MetaCore pathway analysis revealed that such expression alterations of the 208 miRNAs were significantly accumulated in four pathways, which included “Development microRNA-dependent inhibition of epithelial-mesenchymal transition (EMT) pathway” (P = 8.695×10−15). The expression levels of any of 5 EMT-related miRNAs, i.e. miR-200a, miR-200c, miR-141, miR-429 and miR-200b, called as miR-200 family, were reduced in 93 (98%) of the examined 95 RCCs. Quantitative RT-PCR analysis verified that the expression levels of miR-200 family were significantly reduced in T samples relative to the corresponding N samples. Additionally, the mRNA expression levels of the ZEB1 and ZEB2 genes, which were downstream targets of miR-200 family, were significantly increased, and their target CDH1 gene expression was further decrease in T samples. The expression levels of miR-200 family in T samples were significantly correlated with clinicopathological parameters reflecting tumor aggressiveness, such as invasive macroscopic configuration, higher histological grade, vascular involvement, renal vein tumor thrombus, invasive growth pattern, tumor necrosis, distant metastasis and higher histological TNM stage. Moreover, the expression levels of miR-200 family were significantly correlated with both of cancer-free and overall survival rates of patients with clear cell RCCs.

[Conclusions] These data suggested that reduced expression of miR-200 family may participate in the malignant progression resulting in poorer patient outcome of clear cell RCCs, via regulation of the EMT pathway.

Citation Format: Masahiro Gotoh, Eri Arai, Akio Matsuda, Hiroyuki Fujimoto, Kenji Matsumoto, Yae Kanai. Reduced expression of microRNA-200 family associated with aggressiveness and poorer patient outcome of clear cell renal cell carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1065.
Abstract 107: Unsupervised analysis of cancer-cell intrinsic transcriptional traits defines a new classification system for colorectal cancer with improved predictive and prognostic value
Authors: Bertotti Andrea , Isella Claudio , Bellomo Sara E. , Brundu Francesco , Galimi Francesco , Ficarra Elisa , Trusolino Livio , Medico Enzo
Published: July 2016

Recent work built on published transcriptional subtypes of colorectal cancer (CRC) has shown that tumor stromal content heavily impacts CRC classification, with clinical and biological implications. Lineage-dependent transcriptional components of stromal origin could therefore dominate over more subtle, yet relevant, gene expression traits inherent to cancer cells. Based on the notion that in patient-derived xenografts (PDXs) stromal cells of the human tumor are substituted by their murine counterparts, we assessed cancer-cell intrinsic transcriptional features by human-specific expression profiling on 515 PDXs from 244 CRC patients. We identified five CRC intrinsic subtypes (CRIS) endowed with molecular, functional and phenotypic peculiarities, many of which were not previously evidenced: CRIS-A was a MIN-like, KRAS-mutated, and secretory subtype with sustained glycolytic metabolism and inflammatory traits; CRIS-B grouped a subset of poorly differentiated tumors with active TGFβ signaling and EMT features; CRIS-C clustered KRAS wild-type CIN tumors exhibiting elevated EGFR pathway activity and MYC copy number gains; CRIS-D tumors were characterized by a typical stem phenotype with high WNT signaling, associated with strong enrichment in IGF2 amplification/overexpression and FGFR autocrine stimulation; CRIS-E again displayed WNT-related features, but associated with a Paneth-like phenotype and a TP53-mutated genotype. CRIS subtypes successfully categorized independent sets of primary and metastatic CRC tumors, with limited overlap on existing transcriptional classes and unprecedented predictive and prognostic performances. Integration of stroma-centered transcriptional profiles with cancer-cell intrinsic classification by CRIS further enhanced prognostic significance.

Citation Format: Andrea Bertotti, Claudio Isella, Sara E. Bellomo, Francesco Brundu, Francesco Galimi, Elisa Ficarra, Livio Trusolino, Enzo Medico. Unsupervised analysis of cancer-cell intrinsic transcriptional traits defines a new classification system for colorectal cancer with improved predictive and prognostic value. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 107.
Abstract 1081: Activity of microRNA replacement reagent, MRX34, in multiple myeloma in vivo model
Authors: Jung Woo June , Koh Youngil , Kim Sinil , Park Hyejoo , Yoon Sung-Soo
Published: July 2016

Background: Despite recent progress in multiple myeloma (MM) treatment with the advent of new targeted anticancer agents such as Bortezomib, Lenalidomide and, Thalidomide MM still remains incurable. To improve clinical outcome of MM patients, many research have been trying to develop novel targeted drugs. Recent studies demonstrated that microRNA Replacement reagent could be a promising therapeutic strategy in patients with hematologic malignancies. Hence, we tried to examine the efficacy of MRX34, a microRNA-34 replacement reagent, in MM animal model.

Methods: We examined the anti-tumor activity of a MRX34 against 3MM cell lines (LP1, MOLP-8, and U266), and investigated the survival effects through which this novel agent shows anti-MM activity. For an animal study, we used NRG (NOD-Rag1null IL2rgnull, CB-17 SCID) mice that were injected with MM cells via tail vein. MOLP-8, LP1 and U266 cells were injected Intravenous 106 cells of NRG. When tumors were measurable, mice were assigned to 3 treatment groups receiving the vehicle alone (control), MRX34 0.3mg/kg treatment group and, MRX34 1mg/kg treatment group were given Intravenous 5 times a week.

Results: Median survival of mice injected with LP1, MOLP-8 and U266 cell lines were 28days, 29 days and 34days respectively without any treatment. When mice were treated with MRX-34, median survival was 33days, 37days and 42days respectively. There was a significant survival advantage with MRX-34 treatment in MOLP-8 and U266 cell lines. For monoclonal proteins, mice treated with MRX34 group are suppressed M-protein level almost 30% less than M-protein.

Summary / Conclusion: Our data indicated that MRX34 had beneficial effects in multiple myeloma animal model. Mice treated with MRX34 showed M-protein suppression and survival advantage. Our results warrant further clinical studies using this novel agent in MM.

Citation Format: Woo June Jung, Youngil Koh, Sinil Kim, Hyejoo Park, Sung-Soo Yoon. Activity of microRNA replacement reagent, MRX34, in multiple myeloma in vivo model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1081.
Abstract 1082: High-throughput,purification-free, multiplexed profiling of circulating miRNA for discovery,validation, and diagnostics
Authors: Tytell Jessica , Stoner Issac , Tackett Michael , Doran Graeme , Rafferty Conor , Windemuth Andreas , Pregibon Daniel
Published: July 2016

To address the needs for circulating miRNA biomarker validation, we developed the Multiplexed Circulating microRNA assay. This assay enables the detection of up to 68 microRNA targets per sample in 96-well format with readout on standard flow cytometers and analysis with an included bioinformatics software package. The Circulating microRNA assay combines particle-based multiplexing, using patented Firefly hydrogel particles, with single-step RT-PCR signal amplification using universal primers. Thus, the Circulating microRNA assay leverages PCR sensitivity while eliminating the need for separate reverse transcription reactions and mitigating amplification biases introduced by target-specific qPCR. Furthermore, the ability to multiplex targets in each well eliminates the need to split valuable samples into multiple reactions. Results from the Circulating microRNA assay are displayed and interpreted using our included Firefly Analysis Workbench, which allows visualization, normalization, and export of experimental data with only a few mouse clicks. To aid discovery and validation of biomarkers, we have generated fixed panels for Oncology, Cardiology, Neurology, Immunology, and Liver Toxicology. These carefully curated panels include hemolysis markers to assess sample quality, as well as critical normalization factors. Here we present the data from several studies investigating circulating and tumor microRNA profiles using the Firefly Circulating microRNA Assay Fixed Panels.

Together, this novel combination of bioinformatics tools and multiplexed, high-sensitivity assays enables rapid discovery and validation of microRNA biomarker signatures from fluid specimens.

Citation Format: Jessica Tytell, Issac Stoner, Michael Tackett, Graeme Doran, Conor Rafferty, Andreas Windemuth, Daniel Pregibon. High-throughput,purification-free, multiplexed profiling of circulating miRNA for discovery,validation, and diagnostics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1082.
Abstract 1089: Cardiac glycosides affect miRNA expression profiles in renal cell carcinoma cell lines
Authors: Nolte Elke , Wach Sven , da Silva Izabella T. , Ekici Arif , Müller-Uri Frieder , Kreis Wolfgang , Vera-Gonzales Julio , Wullich Bernd , Taubert Helge , Lai Xin
Published: July 2016

Background: Renal Cell Carcinoma (RCC) is the ninth most common cancer worldwide, with about 338,000 new cases diagnosed in 2012. Especially metastatic renal cell carcinoma has still despite surgical cytoreduction, VEGF-tyrosine kinase inhibitors, mTOR inhibitors and first promising results of immunotherapies a very poor prognosis what implies an urgent need for additional therapies. Cardiac glycosides have come into the focus as potential cancer treatment option. They can have anti-proliferative and apoptotic characteristics in several cancer cell lines but affect only in a much lesser extent normal cell lines. MicroRNAs are small non-coding RNAs of 17-25 nucleotides length that widely exist and they play an important regulating role in many normal and pathophysiological cellular processes, as cell proliferation, differentiation, induction of apoptosis, tumorigenesis and tumor progression.

Material and Methods: Four renal cell carcinoma cell lines were treated with three different cardiac glycosides in the IC50 concentrations during 72h. For the investigation of miRNA expression miRNA microarray expression analyses were performed. In silico target gene and pathway enrichment analyses were performed.

Results: An overlap between all treatments and all cell lines could be detected for two mature miRNAs and two premature miRNAs. Pathway enrichment analysis identified MAPK and axon guidance pathways. Moreover, we looked for genes predicted to be regulated by the deregulated miRNAs in all cell lines at all treatments in the MAPK and the axon guidance pathways.

Conclusion: The identification and characterization of deregulated miRNAs after cardiac glycoside treatment could give insight in their gene regulation and thus help to clarify the molecular basis of their anti-proliferative and apoptotic effects.

Citation Format: Elke Nolte, Sven Wach, Izabella T. da Silva, Arif Ekici, Frieder Müller-Uri, Wolfgang Kreis, Julio Vera-Gonzales, Bernd Wullich, Helge Taubert, Xin Lai. Cardiac glycosides affect miRNA expression profiles in renal cell carcinoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1089.
Abstract 1090: microRNA regulation of Nrf2 and the antioxidant response in breast cancer cells following redox therapy
Authors: Mascia Francesca , Pokrzywinski Kaytee L. , Rao. V. Ashutosh
Published: July 2016

Mitoquinone (MitoQ), a redox-active mitochondrially targeted chemotherapy, activates autophagy through the production of reactive oxygen species (ROS) in breast cancer cells. Studies have also shown a concomitant activation of Nrf2 and the antioxidant response with MitoQ treatment. Nrf2 (nuclear factor (erythroid-derived 2)-like 2) is a transcription factor that regulates the antioxidant response element. Drugs that stimulate Nrf2 are ideal for the treatment of diseases induced by oxidative stress; however, over-activation of Nrf2 may promote tumor growth and diminish the effects of redox active chemotherapies. Therefore, understanding the relationship between autophagy and oxidative stress is crucial for determining the fate of cancer cells exposed to redox-active oncology agents. The aim of this study was to evaluate microRNA (miRNA) as a link between oxidative stress and autophagy through modulation of Nrf2 activity. microRNA are endogenous small (18-22 base pairs long) double stranded RNA that bind to the 3’ UTR of target mRNA. miRNA function through translational repression via mRNA recycling and degradation. To this aim we set out on finding miRNA that suppress the antioxidant response, modulated by Nrf2, in cancer cells after redox therapy with the goal of inducing a death response. After 24 hours of non-target pool or Nrf2 silencing, using small interfering RNAs, MDA-MB-231 cells were treated with DMSO or MitoQ for 18 hours. RNA was extracted and miRNAs were identified using Illumina RNA sequencing. DEseq revealed 97 miRNAs that were differentially expressed at α < 0.05. Biologically significant miRNAs were then distinguished by using a Log2 fold change cut off of -1.0 < and < 1.0, which identified 39 microRNAs. Since miRNA should be high when their target is low we were predominantly interested in the 15 miRNAs that were down-regulated (DMSO:MitoQ). 7 of the 15 down-regulated miRNAs were also down-regulated in the presence of Nrf2 silencing in both the DMSO and MitoQ treatments. We are currently investigating these 7 miRNAs in terms of the specific effect they have on the Nrf2 pathway during redox therapy.

Citation Format: Francesca Mascia, Kaytee L. Pokrzywinski, V. Ashutosh Rao. microRNA regulation of Nrf2 and the antioxidant response in breast cancer cells following redox therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1090.
Abstract 1093: The melanoma-enriched microRNA miR-4731 regulates genes involved in cell cycle and the melanosome
Authors: Stark Mitchell S. , Tom Lisa , Boyle Glen M. , Bonazzi Vanessa F. , Herington Adrian C. , Pollock Pamela M. , Hayward Nicholas K.
Published: July 2016

We previously identified miR-4731-5p (miR-4731) as a melanoma-enriched microRNA following comparison of melanoma with other cell lines from solid malignancies. Additionally, miR-4731 has been found in serum from melanoma patients and expressed less abundantly in metastatic melanomas from stage IV patients relative to stage III patients. As miR-4731 has no known function, we used biotin-labelled miRNA duplex pull-down to identify binding targets of miR-4731 in three melanoma cell lines. Using the miRanda miRNA binding algorithm, all pulled-down transcripts common to the three cell lines (n = 1092) were predicted to be targets of miR-4731 and gene-set enrichment analysis of these (via STRING v9.1) highlighted significantly associated genes related to the ‘cell cycle’ and ‘melanosome’ pathways. Following miR-4731 overexpression, a selection (n = 81) of pull-down transcripts underwent validation using a custom qRT-PCR array. These data revealed that miR-4731 regulates multiple genes associated with the cell cycle (e.g. CCNA2, ORC5L, and PCNA) and melanosome (e.g. RAB7A, CTSD, and GNA13). Furthermore, members of the synovial sarcoma X breakpoint family (SSX) (melanoma growth promoters) were also down-regulated (e.g. SSX2, SSX4, and SSX4B) as result of miR-4731 overexpression. We therefore speculate that loss of miR-4731 expression supports melanoma growth by, in part; reducing its regulatory control of SSX expression levels together with members of the cell cycle pathway, which warrants further investigation.

Citation Format: Mitchell S. Stark, Lisa Tom, Glen M. Boyle, Vanessa F. Bonazzi, Adrian C. Herington, Pamela M. Pollock, Nicholas K. Hayward. The melanoma-enriched microRNA miR-4731 regulates genes involved in cell cycle and the melanosome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1093.
Abstract 1096: microRNA-193a-3p acts as a tumor suppressor in BRAF-mutated colorectal cancer
Authors: Takahashi Hidekazu , Takahashi Masanobu , Takahashi Shin , Shimodaira Hideki , Ishioka Chikashi
Published: July 2016

The aim of this study was to identify microRNAs (miRNAs) specifically dysregulated in BRAF-mutant colorectal cancer, a malignant subtype of colorectal cancer. The genome-wide miRNA expression analysis in a screening set (n = 30) identified 22 miRNAs specifically dysregulated in BRAF-mutant cancers. Among the top 5 dysregulated miRNAs, the validation analysis using another set of patients (n = 34) confirmed miR-31 and miR-135b as significantly up-regulated miRNAs and miR-193a-3p as a significantly down-regulated one. Moreover, functional assays supported its tumor-suppressive role in colorectal cancer. Finally, the expression status of miR-193a-3p correlated with clinical outcome of patients treated with anti-EGFR antibodies. Taken together, these results show a novel functional role of miR-193a-3p as a tumor suppressive miRNA in colorectal cancer, particularly with BRAF mutations. Further studies are needed to clarify the precise molecular mechanisms as to how this miRNA is involved in colorectal tumorigenesis, which could help further develop a therapeutic strategy for colorectal cancer.

Citation Format: Hidekazu Takahashi, Masanobu Takahashi, Shin Takahashi, Hideki Shimodaira, Chikashi Ishioka. microRNA-193a-3p acts as a tumor suppressor in BRAF-mutated colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1096.
Abstract 1097: The miR-17-92 microRNA cluster plays a crucial role in osteosarcoma progression
Authors: Varshney Jyotika , Slipek Nicholas J. , Osborne John , Chang Adrian , Sarver Anne E. , Cornax Ingrid , O’Sullivan Gerry M. , Subramanian Subbaya , Largaespada David A.
Published: July 2016

Osteosarcoma is the most common primary bone malignancy that affects adolescents. Around 30% of patients with localized osteosarcoma and 70% of patients with metastasis will experience treatment failure within 5 years of diagnosis. The complex biology of osteosarcoma and astounding genetic heterogeneity has made it challenging to identify effective new gene targets and therapeutic agents. Our studies found an overall overexpression of a microRNA cluster, miR-17-92 microRNAs in human primary osteosarcoma compared to normal bone. We learned that upregulation of this miR-17-92 cluster simultaneously silences a suite of key tumor suppressors. Using data from a novel spontaneous osteosarcoma mouse model and genetic screen, we discovered miR-17-92 targets, such as PTEN, PTRPD and SRGAP2, which are potential tumor suppressor genes. Specifically blocking miR-17-92 function in osteosarcoma cells reduced their migration and ability to grow larger tumors in immunodeficient mice compared to the controls. Also, knockdown of miR-17-92 cluster microRNAs led to increase in the levels of PTPRD and SRGAP2 in cells as well as tumors; further suggesting that miR-17-92 is targeting these genes. We also performed gain-of-function of miR-17-92 studies in a poorly aggressive osteosarcoma cell line and found that overexpression of miR-17-92 leads to ability to grow in an anchorage independent manner and form tumors in immunodeficient mice, both features that are lacking in the parental line. Ongoing RNA sequencing studies on miR-17-92 target transcripts in osteosarcoma cells, and functional analyses of miR-17-92 deletion mutants, will be presented. In an attempt to target miR-17-92 miRNA expression, we have tested small molecules. Our data suggests that triptolide, a diterpenoid epoxide, inhibits MYC expression and downregulates miR-17-92 miRNAs resulting in upregulation of several tumor suppressor driver proteins including PTEN, PTPRD, and SRGAP2. Together, our data suggests that upregulation of miR-17-92 miRNAs contributes to osteosarcoma progression and triptolide inhibits miR-17-92 expression. These data have implications for how sarcomas develop in general and suggest a new way to treat cancer by targeting microRNAs using small molecules.

Citation Format: Jyotika Varshney, Nicholas J. Slipek, John Osborne, Adrian Chang, Anne E. Sarver, Ingrid Cornax, Gerry M. O’ Sullivan, Subbaya Subramanian, David A. Largaespada. The miR-17-92 microRNA cluster plays a crucial role in osteosarcoma progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1097.
Abstract 1115: Serum miR152 as a novel prognostic biomarker and potential tumor suppressor for early stage non-small cell lung cancer
Authors: Oba Taro , Ye Yuanqing , Zhang Liren , Lin Jing , Gentile Emanuela , Wang Jing , Zhao Yang , Gu Jiang , Wistuba Ignacio , Roth Jack A. , Wu Xifeng , Ji Lin
Published: July 2016

Cancer-derived circulating miRNAs represent a promising new class of circulating biomarkers for cancer detection, prognosis, and therapeutic targets. We analyzed the differential c-miRNA expression levels of ∼100 candidate serum miRNAs by quantitative RT-PCR in 174 Caucasian, early stage lung cancer patients and 184 Caucasian healthy controls. Serum miR152 exhibited significant differential expression between cases and controls (P = 0.014). Low levels of serum miR152 expression were consistently associated with an increased risk of lung cancer. The down-regulated miR152 expression was also detected across a panel of 40 NSCLC and normal bronchial epithelial cell lines and in 250 primary tumors by miRNA expression profiling on microarrays. The tumor-derived origin of the c-miR150 was confirmed by an increased level of serum miR152 with increased tumor volumes three weeks after tumor cell inoculation in a human H1299 tumor xenograft mouse model by qRT-PCR assay. Ectopic expression of miR-152 suppressed tumor cell proliferation, colony formation, and cell migration/invasion in NSCLC H1299, H460, and A549 cells transfected by miR-152 expression plasmids. Inhibition of miR-152 expression by a miR-152 inhibitor promoted tumor cell proliferation in HCC15 cells. Overexpression of miR152 in H1299, H460 and A549 cells significantly down-regulated the constitutive expression of the DNMT1 miR-152 target gene and protein, suggesting its potential role in the epigenetic regulation of lung cancer pathogenesis. These results suggest that serum miR-152 may serve as a novel negative predictor for lung cancer risk, recurrence, and survival in early stage NSCLC and function as a potential tumor suppressor miRNA by epigenetic down-regulation of oncogenesis.(This study is partially supported by NIH/NCI grants Lung SPORE 5P50CA070907 and R01CA176568, a CPRIT Grant and a MDACC Moonshot Program Grant)

Citation Format: Taro Oba, Yuanqing Ye, Liren Zhang, Jing Lin, Emanuela Gentile, Jing Wang, Yang Zhao, Jiang Gu, Ignacio Wistuba, Jack A. Roth, Xifeng Wu, Lin Ji. Serum miR152 as a novel prognostic biomarker and potential tumor suppressor for early stage non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1115.
Abstract 1116: Contribution of miR-199a to subtype switching of the SW13 adrenocortical carcinoma cell line
Authors: Gerona Tracy , Davis McKale , Hull Elizabeth
Published: July 2016

The human adrenocortical carcinoma SW13 cell line has two subtypes that are phenotypically distinct. The SW13- subtype is highly proliferative and epithelial-like, while the SW13+ subtype is slow growing, motile, and mesenchymal-like. These differences are mediated in part by the post-transcriptional regulation of BRM, which is expressed in SW13+ cells, and not detectable in SW13- cells. While treatment with histone deacetylase inhibitors (HDACi) can force cells to transition from SW13- to SW13+, the epigenetic mechanisms involved in the subtype switch are unknown. Recently, it was shown that miR-199a is a potent negative regulator of BRM expression in many epithelial tumor cell lines. Given this, we examined the expression of miR-199a in the two SW13 subtypes, and indeed found that miR-199a expression was significantly higher in the BRM-deficient SW13- cells than the BRM-expressing SW13+ cells. To begin to further assess the involvement of miR-199a in the SW13 subtype switch, SW13+ and SW13- cells were treated with either a miR-199a mimic or miR-199a inhibitor, respectively and assessed for differences in BRM mRNA and protein expression, as well changes in cell-type morphology. Our results indicate that although miR-199a can mediate differences in BRM expression, miR-199a alone is not sufficient to induce a full subtype conversion as evidenced by retention of subtype morphology following miR-199a inhibition and over-expression. We are currently investigating the role of miR-199a in HDACi mediated subtype conversion, as well as the expression of other miRNA and their targets that are differentially expressed between the two SW13 subtypes.

Citation Format: Tracy Gerona, McKale Davis, Elizabeth Hull. Contribution of miR-199a to subtype switching of the SW13 adrenocortical carcinoma cell line. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1116.
Abstract 1121: Validation of FOXR2 and ARHGAP36 as oncogenes in medulloblastoma
Authors: Jackson Pauline , Larsson Alex , Das Paramita , Largaespada David
Published: July 2016

We are working to validate two putative oncogenes in medulloblastoma (MB), ARHGAP36 and FOXR2. These genes were identified as MB drivers using a Sleeping Beauty mutagenesis screen in mice and are overexpressed in subsets of human MB. We are using gain (GOF) and loss of function (LOF) studies both in vitro and in vivo to understand the roles of FOXR2 and ARHGAP36 in MB genesis. In GOF studies, both genes individually drove anchorage independent growth in a mouse cerebellar progenitor cell line (C17.2) and a human MB cell line (ONS76). When overexpressed individually in C17.2 cells, both FOXR2 and ARHGAP36 drove tumor formation in the flank of NU/J mice. We are also using an orthotopic injection model with C17.2 cells overexpressing either ARHGAP36 or FOXR2 individually to determine if these genes drive tumor formation in a more relevant setting. RNA sequencing and reverse-phase protein array analyses were also performed on WT and C17 cells overexpressing each oncogene to identify their effects in an unbiased manner. We are working to validate those effects using RT-PCR and Western analysis. In LOF studies, the CRISPR/Cas system is being used to create mutations in either the ARHGAP36 or FOXR2 locus in human MB cell lines (ONS76, MED8A, Daoy). This has been accomplished with ARHGAP36 in ONS76 cells, and we are currently characterizing those cells using proliferation, soft agar colony formation, and tumor formation assays. Lastly, we are working to create GOF and LOF mouse models of both FOXR2 and ARHGAP36 to characterize their oncogenic potential in vivo. We identified ARHGAP36 and FOXR2 as putative oncogenes in MB and our findings may elucidate novel targets for therapeutic efforts aimed at treating patients with MB.

Citation Format: Pauline Jackson, Alex Larsson, Paramita Das, David Largaespada. Validation of FOXR2 and ARHGAP36 as oncogenes in medulloblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1121.
Abstract 1122: FGFR1 can act as an oncogene or a tumor suppressor depending on the molecular context
Authors: Quintanal-Villalonga Álvaro , Ojeda-Márquez Laura , Marrugal Ángela , Suárez Rocío , Ferrer Irene , Carnero Amancio , Molina-Pinelo Sonia , Paz-Ares Luis
Published: July 2016

FGFR family of proteins has been extensively related to oncogenic properties in several types of tumors, including lung cancer. Among its members, FGFR1 has been related to squamous cell lung carcinoma, where FGFR1 amplification is present in 20% of patients. However, its role in lung cancer has not yet been thoroughly described.

Several lung cancer cell lines with different genetic backgrounds were transfected with plasmids to either overexpress or silence FGFR1. Then several tumorigenic abilities were tested in vitro and in vivo. mRNA from Paraffin-embedded tissue of a cohort of lung cancer patients was extracted and FGFR1 expression levels were measured and related to clinical characteristics.

FGFR1 increases oncogenic properties in SCC cell lines, but exerts anti-oncogenic effects in several lung ADC cell lines, suggesting a tumor suppressor role under certain circumstances. This is a consequence of differentially expressed genes among these two histologies. According to this, analysis of FGFR1 mRNA expression of a cohort of lung cancer patients revealed that high FGFR1 mRNA expression was associated to a shorter overall survival (OS) and progression free survival (PFS) in lung SCC patients. However, a trend for longer OS was observed for the ADC patients with higher FGFR1 mRNA expression.

FGFR1 has the potential to be either a tumor suppressor or an oncogene and the molecular context is a determining factor to define its final role in tumorigenesis.

Citation Format: Álvaro Quintanal-Villalonga, Laura Ojeda-Márquez, Ángela Marrugal, Rocío Suárez, Irene Ferrer, Amancio Carnero, Sonia Molina-Pinelo, Luis Paz-Ares. FGFR1 can act as an oncogene or a tumor suppressor depending on the molecular context. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1122.
Abstract 1139: Translational control of MYC protein stability as a target of small molecules
Authors: Ikegaki Naohiko , Baloch Luqman , Chu Christopher , Lomahan Joshua , Harris Jamie , Chiu Bill
Published: July 2016

It is well established that MYC family proteins drive tumor growth and sustain the malignant phenotype. However, MYC family proteins have gained the reputation of being “un-druggable” by small molecule inhibitors. This is because MYC proteins lack intrinsic enzymatic activities or appropriate molecular grooves to which small molecules may bind and inhibit their function. Nonetheless, previous studies suggest that MYC family proteins (MYC, MYCN, MYCL) can be “targetable” indirectly by accelerating their turnover rate at different biochemical pathways that regulate the steady state level of the proteins. These include transcriptional-inhibition of the corresponding genes and acceleration of their proteasome-dependent degradation. The goal of this study is to gain a better understanding of how the MYC family protein turnover is regulated, which in turn might help develop future new drugs against “MYC family-driven” cancers. We have used neuroblastoma, a childhood cancer, as a disease model and identified several small molecules that rapidly destabilize MYC and MYCN proteins in MYC-driven neuroblastoma cells. The small molecules include FCCP, OSU-03012 and Salinomycin. Preliminary data suggest that the common target of these compounds is mitochondria. Recently, we have also found Halofuginone, an FDA approved orphan drug for Scleroderma treatment exhibits a similar MYC-destabilizing activity. At submicromolar concentrations and less than one hour of drug-treatment, Halofuginone rapidly causes significant down-regulation of MYC proteins in neuroblastoma cells. Halofuginone is known to activate the eIF2α pathway, which controls translation. Activation of the eIF2α pathway is initiated by the activation of eIF2a kinases (GCN2, HRI, PERK, PKR) via autophosphorylation by various stress-related stimuli such as reactive oxygen species and misfolded proteins. The activated eIF2α kinases then inactivates eIF2α by phosphorylation, which subsequently halts translation globally. Interestingly, FCCP, OSU-03012 and Salinomycin treatments also caused eIF2α phosphorylation. These observations suggest that activation of the eIF2α pathway results in translational block of the MYC family protein expression in tumor cells. Because half-lives of MYC family proteins are very short, a sudden attenuation in protein translation can cause rapid down-regulation of the proteins. Hence, activation of the eIF2α pathway by small molecules may prove effective for rapid down-regulation of MYC family proteins in MYC-driven cancer cells.

Citation Format: Naohiko Ikegaki, Luqman Baloch, Christopher Chu, Joshua Lomahan, Jamie Harris, Bill Chiu. Translational control of MYC protein stability as a target of small molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1139.
Abstract 1153: Hand-1 overexpression inhibits medulloblastoma cell invasion and metastatic ability via Oct-3/4 / β-catenin interaction
Authors: Asuthkar Swapna , Guda Maheedhara R. , Tsung Andrew J. , Antony Reuben , Dinh Dzung H. , Velpula Kiran K.
Published: July 2016

Medulloblastoma (MB) is the most common malignant brain tumor in children. Treatment of medulloblastoma includes surgery, radiation and chemotherapy. Despite long-term survival for the majority of children, 50% of medulloblastomas recur. Recently, medulloblastomas are classified into four distinct molecular subgroups, viz., WNT, SHH, group 3, and group 4 based on constitutive activation of WNT/β-catenin, SHH, and NOTCH signaling pathways that regulate cancer stem cell pluripotency, tumor growth, progression and metastasis. Previously, we investigated the molecular machinery underlying uPAR and WNT/β-catenin interdependent signaling pathways in medulloblastoma stem cells. Further, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. Complementing to this we demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand-1. Here, we show that Hand-1 expression is observed to be downregulated in all the molecular subgroups of medulloblastoma. We also showed that the pluripotency marker, Oct-3/4 (encoded by the Pou5f1 gene) interacts with β-catenin and Hand-1 that ultimately regulated the uPAR expression. Our preliminary study demonstrated that Oct-3/4 preferentially cooperated with β-catenin in medulloblastoma to maintain tumor progression. Immunohistostaining of clinical specimens also confirmed our aforementioned results. Taken together, our studies show that overexpression of Hand-1 in medulloblastoma cells may effectively inhibit uPAR-induced invasion and metastatic ability, thereby regulating Oct-3/4/β-catenin balance.

Citation Format: Swapna Asuthkar, Maheedhara R. Guda, Andrew J. Tsung, Reuben Antony, Dzung H. Dinh, Kiran K. Velpula. Hand-1 overexpression inhibits medulloblastoma cell invasion and metastatic ability via Oct-3/4 / β-catenin interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1153.
Abstract 1166: The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) invasion and migration
Authors: Hou Hsin-Han , Yu Chong-Jen
Published: July 2016

Emerging evidence has indicated that proteins of a disintergrin and metalloprotease (ADAM) family contribute to cancer progression and metastasis. One member of this family, ADAM15, has been shown to be upregulated in multiple cancers, including gastric, lung, breast, and prostate cancers, and the enzymatic activities of its extracellular metalloprotrease domain promote breast cancer proliferation and migration through mediating ErbB signaling pathway. The patients with ADAM15 high-expressing lung tumors have shorter survival time and enzymatic activity of ADAM15 extracellular domain activates MMP-9 and promotes NSCLC migration and invasion. We firstly demonstrated other than extracellular enzymetic activity, the longest isoform of ADAM15 (ADAM15 i6), which contains the most cytoplasmic Src homology 3 (SH3) binding motifs, significantly upregulated in primary lung cancer tissues and promoted NSCLC proliferation via growth factor receptor-bound protein 2 (Grb2) and Src homolog 2 domain containing (Shc) association. In this study, we further explore the roles of ADAM15 cytosolic domain in NSCLC invasion and migration. Overexpression of ADAM15 i6 promoted CL1-0 cell invasion and migration according to the transwell and wound healing assay. Ablation of Ras protein activator (RASA)1 and protein tyrosine kinase (PTK)6 attenuated the ADAM15 i6-promoted invasion and migration respectively. Thus, we identified a novel mechanism of the ADAM15 cytoplasmic domain in NSCLC tumor progression, which will shed light on the molecular mechanisms of ADAM proteins, and facilitate development of novel therapy in NSCLC.

Citation Format: Hsin-Han Hou, Chong-Jen Yu. The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) invasion and migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1166.