bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1317: Simultaneous identification of candidate melanoma risk variants using massively parallel reporter assay
Authors: Choi Jiyeon , Zhang Tongwu , Kovacs Michael , Xu Mai , Lam Nghi , Colli Leandro , Brown Kevin
Published: July 2017

Recent melanoma Genome-Wide Association Study (GWAS) has identified 20 common susceptibility loci via meta-analysis of ~15,000 cutaneous malignant melanoma cases. While GWAS is extremely powerful in identifying genomic regions contributing to melanoma risk, teasing out functional variants and unraveling molecular mechanisms by which the risk is conferred presents a tremendous challenge due to limited resolution of genetic structure and lack of high-throughput assay system. To simultaneously identify functional risk variants from multiple GWAS loci, we employed recently developed Massively-Parallel Reporter Assays (MPRA). Based on the hypothesis that many GWAS functional variants regulate gene expression through transcriptional mechanism, we systematically examined allele-specific transcriptional activities of genetically indistinguishable candidate variants. Out of ~2,800 melanoma-associated variants (r2>0.4, EUR) from 16 melanoma loci for which transcriptional mechanism could be applied, 835 variants were prioritized based on their relevance as melanocyte-/melanoma-specific cis-element represented by ENCODE annotation as open chromatin or putative promoter/enhancer histone marks in melanocytes and melanoma cell lines. Pooled oligoes of 145bp encompassing each variant with either melanoma risk or protective allele coupled with 10 different unique sequence tags were cloned into luciferase vectors and transfected to melanoma cell lines. Resulting expressed tag counts were subsequently determined using massively parallel sequencing. We were able to identify ~200 potentially functional variants displaying allelic transcriptional activity by combining transfections in a melanoma cell line and HEK293FT cell line. Top functional SNPs from melanoma GWAS loci were also shown as significant eQTL SNPs in human melanocytes including MX2 on chromosome 21. Overlaying MPRA functional SNPs with melanocyte eQTL SNPs further enabled isolation of a functional risk variant among several highly linked MX2 eQTL SNPs. The results from this analysis will greatly accelerate the identification of functional melanoma risk variants and further shed light on molecular mechanisms of genetic susceptibility of melanoma in population.

Citation Format: Jiyeon Choi, Tongwu Zhang, Michael Kovacs, Mai Xu, Nghi Lam, Leandro Colli, Kevin Brown. Simultaneous identification of candidate melanoma risk variants using massively parallel reporter assay [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 1317. doi:10.1158/1538-7445.AM2017-1317
Abstract 132: African polyherbal formulation alleviates benzene-induced leukemia in Wistar rats
Authors: Akanni Olufemi E. , Faremi Ayodeji , Agboola Aminat O. , Akanni Adekemi R. , Bamisaye Oluseyi E.
Published: July 2017

Background: Chemotherapy and radiotherapy are effective cancer treatment options but they are accompanied by serious side effects. Therefore, more effective therapies are sorely needed. This study investigated the chemotherapeutic effects of a polyherbal formulation on benzene induced leukemia in Wistar rats. The polyherbal formulation is composed of fruits such as Orange, Lime, Lemon, Pineapple, Grape and Vegetables like; Pumpkin leave, Garden egg in honey medium.

Method: Leukemia was induced by injecting 0.2 ml of benzene solution intravenously through the tail at 48 h intervals for four weeks. Leukemia developed in 92% of rats within 2 to 4 weeks after the last benzene injection and further observed for leukemia development in appropriate rat groups. Leukemia burden was assessed using indicator parameters such as total leukocyte, red blood cell count, hematocrit, and hemoglobin concentration. Immunophenotyping enumeration of CD 19 expression also corroborated the leukemia assessment by providing specific cellular marker for differentiation of the developed leukemia type. Exactly 0.48 ml of the polyherbal formulation was administered orally by gavage using oral cannular once daily post leukemia induction for four weeks.

Results: Leukemia induction reflected in significant reduction in hematocrit, hemoglobin concentration, red cell count and a marked and a significant increase in leukocyte count and CD 19 expression over the control (p < 0.05). There is significant difference between the leukemia group treated with polyherbal formulation and the leukemia positive control group untreated.

Conclusion: This study reveals profound chemotherapeutic activity of the polyherbal formulation against experimentally developed leukemia by reversing the induced leukemia in the positive control rat group, hence the ability of the polyherbal formulation in alleviating the cancer symptoms.

Table 1.Hematological parameters in polyherbal formulation treated leukemia bearing Wistar rats.Estimated parametersNegative control rats (Mean±SD)Leukemia Positive rats. (Mean±SD)Polyherbal formulation treated leukemic rats (Mean±SD)CD 19 (X 109/L)47.59±1.3989.41±1.7262.32 + 1.42*Total Leukocyte count (X 109/L)6.84 + 0.3011.08 + 0.317.45 + 0.65*Red blood cells(X 1012/L)6.30 + 0.383.5 + 0.385.10 + 0.03*Hematocrit (L/L)0.43 + 0.170.26. + 0.200.37 + 0.08*Hemoglobin (g/dl)13.62 + 0.696.63 + 0.9611.80 + 2.96*Platelets (X 109/L)554.80 + 92.23389.50 + 146.10425.10 + 60.8**Significance at p<0.05

Citation Format: Olufemi E. Akanni, Ayodeji Faremi, Aminat O. Agboola, Adekemi R. Akanni, Oluseyi E. Bamisaye. African polyherbal formulation alleviates benzene-induced leukemia in Wistar rats [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 132. doi:10.1158/1538-7445.AM2017-132
Abstract 1321: Transcriptome-wide association study of prostate cancer risk
Authors: Chen Maxine , Gusev Alexander ,   The PRACTICAL Consortium, Loda Massimo , Mucci Lorelei A. , Stampfer Meir J. , Kraft Peter , Penney Kathryn L.
Published: July 2017

Though more than 150 single nucleotide polymorphisms (SNPs) associated with prostate cancer have been identified through GWAS, we still do not understand the functional consequences of common genetic variation in this disease. Genetic variants may influence complex diseases like prostate cancer through gene expression. However, very large studies evaluating gene expression in prostate cancer are currently infeasible due to the limited availability of prostate cancer tissue and expense of collecting expression data. Alternative approaches have been developed to identify expression-trait associations in study populations without directly measured expression data. One such approach (Gusev et al. Nat Genet. 2016) uses a set of reference individuals with both gene expression and SNP data to develop genetic prediction models of gene expression values. These models are then used to impute cis genetic components of gene expression for a much a larger set of individuals with only genotype and phenotype information. We used Affymetrix gene expression microarray data from normal prostate and prostate tumor tissue and genotype data from prostate cancer patients in the Health Professionals’ Follow-up Study and the Physicians’ Health Study (N=187) as a reference panel for creating the predicted models of the genetic components of expression. We imputed gene expression values into Oncoarray GWAS summary statistics from the PRACTICAL consortium (N ~ 75,000) to assess the association between predicted expression and prostate cancer risk. We present the results of our transcriptome-wide association study in normal prostate and prostate tumor tissue, identifying genes significantly associated with prostate cancer risk after correcting for multiple testing. These genes may provide insight into how genetic variants influence prostate cancer through their effect on gene expression.

Citation Format: Maxine Chen, Alexander Gusev, The PRACTICAL Consortium, Massimo Loda, Lorelei A. Mucci, Meir J. Stampfer, Peter Kraft, Kathryn L. Penney. Transcriptome-wide association study of prostate cancer risk [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 1321. doi:10.1158/1538-7445.AM2017-1321
Abstract 1328: Mechanism of transcriptional regulation of metabotropic glutamate recepter-5 induced by the CXCR4 signaling pathway
Authors: Kuribayashi Nobuyuki , Uchida Daisuke , Kinouchi Makoto , Izumi Sayaka , Kuribayashi Kyoko , Kawamata Hitoshi
Published: July 2017

We have previously demonstrated that stromal cell-derived factor (SDF)-1/CXCR4 system enhances the metastases of oral cancer cells via induction of mGluR5. In this study, we examined the transcriptional regulation of mGluR5 induced by the SDF-1/CXCR4 system, using oral cancer cells, B88, which express functional CXCR4 and have highly metastatic potentials. mGluR5 was markedly induced both by the exogenous SDF-1 addition and overexpression of SDF-1 in B88 cells. These induction was completely inhibited by a MEK inhibitor, U0126, and partially inhibited by a PI3K inhibitor, wortmannin. Furthermore, these inhibitors significantly inhibited the SDF-1/CXCR4 dependent cell-migration in the presence or absence of mGluR5 agonist, DHPG. Recently, several investigators demonstrated that activation of the ERK1/2 pathway is responsible for the transcription of several cancer-associated microRNAs (miRNAs). Thus, we performed miRNA microarray in SDF-1 stimulated B88 cells. we isolated miR-30 family which has predictive binding sites in 3'-UTR of mGluR5 mRNA in silico analysis. We now analyze the regulation of these miRNA by MEK/ERK1/2 and PI3K/Akt pathway.

Citation Format: Nobuyuki Kuribayashi, Daisuke Uchida, Makoto Kinouchi, Sayaka Izumi, Kyoko Kuribayashi, Hitoshi Kawamata. Mechanism of transcriptional regulation of metabotropic glutamate recepter-5 induced by the CXCR4 signaling pathway [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 1328. doi:10.1158/1538-7445.AM2017-1328
Abstract 1329: Inhibition of papillary thyroid cancer cells progression by targeting skp2 via ros-erk-chop-dr5 pathways
Authors: Poyil Pratheeshkumar , Bu Rong , Siraj Abdul K. , P Sandeep Kumar , Al-Kuraya Khawla S.
Published: July 2017

S-phase kinase protein 2 (SKP2), is an F-box protein with proteasomal properties and has been found to be overexpressed in a variety of cancers. However its role in papillary thyroid cancer (PTC) has not been elucidated. Therefore, inactivation of SKP2 could be a viable strategy for the treatment of PTC. In the present study, we investigated the role of SKP2 and its ubiquitin-proteasome pathway in PTC using a tissue microarray cohort of 1022 PTC samples, PTC cell lines and Nude mouse model. Our immunohistochemistry data showed that SKP2 was over-expressed in 75.1% of PTC cases and was clinically, significantly associated with extra thyroidal extension (p=0.0331), Tall cell variant (p=0.0070), and presence in surgical margins (p=0.0347). Bortezomib as well as SKP2 specific siRNA caused downregulation of SKP2 leading to dose-dependent growth inhibition and induction of apoptosis via mitochondrial apoptotic pathway in PTC cell lines. Furthermore, we found that treatment of PTC cells with Bortezomib caused up-regulation of DR5 via generation of reactive oxygen species (ROS). Finally, Bortezomib treatment augmented TRAIL mediated anti-cancer effect on PTC xenograft tumor growth in nude mice. These data suggest that Bortezomib is a viable therapeutic option for the treatment of PTC either alone or in combination with other apoptotic agents such as TRAIL.

Note: This abstract was not presented at the meeting.

Citation Format: Pratheeshkumar Poyil, Rong Bu, Abdul K. Siraj, Sandeep Kumar P, Khawla S. Al-Kuraya. Inhibition of papillary thyroid cancer cells progression by targeting skp2 via ros-erk-chop-dr5 pathways [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 1329. doi:10.1158/1538-7445.AM2017-1329
Abstract 133: Aloe-emodin as a potential lead targets lung cancer stem cells
Authors: Lee Pei-Jung , Chen Wan-Jiun , Ho Chao-Chi , Yang Pan-Chyr , Chen Huei-Wen
Published: July 2017

Cancer stem cells (CSCs) have been proposed to be responsible for tumor initiating, drug resistance, metastasis, and recurrence. Many novel therapeutic strategies have been designed to target and eliminate CSCs. According to our previous study, we have established a model of CSCs and cancer associated fibroblasts (CAFs) co-culture system for anti-CSCs drug screening. Here, we report one of the potential hits screened via this platform and the anti-CSCs activity was further investigated both in vitro and in vivo. Human lung CSCs and CAFs were primary cultured from patient with lung adenocarcinoma according to our previous study. Image-based high content screening system was used to analyze different parameters after drug treatment. Tumorogenicity and self-renew ability are examined by sphere forming ability. Aldehyde dehydrogenase (ALDH) activity was used to analyze stem cell population by flow cytometry. The expression level of stemness-related genes, Nanog, Oct3/4 and Sox2 were validated by real-time reverse transcriptase Q-PCR. The efficacy of the lead on tumor growth was examined by the xenograft model. Lung cancer stemness markers of the xenograft tumor tissues were also evaluated by immunohistochemistry. Using the image-based high content screening system to screen over one thousands of compounds, we have identified aloe-emodin (AE) shows higher potency targeting on lung cancer stem cells (under the concentration of 1 μM) and high specificity targeting on the cancer cell lines (IC50 < 20 μM); compared to normal human bronchial epithelium cells and human normal fibroblast represented by IC50 (26.77 μM v.s. 39.13 μM). The level of stemness markers, Nanog, Sox2 and Oct3/4 were significantly down regulated after AE treatment compared to cisplatin treatment. AE could suppress tumor initiating abilities and self-renew capacities represented by the ability to grow as tumors spheres in CL152 ALDH+ cells. Besides, AE could inhibit ALDH population in CL152 cells (40% reduced). Also, the AE can inhibit the cisplatin-induced ALDH population as well. Furthermore, we found that AE combine with cisplatin could inhibit tumor growth as comparing to cisplatin treatment in subcutaneous models in NOD/SCID mice, whereas, AE can inhibit the level of Nanog in mice tumor tissues. According to these results, AE is a potential lead targeting on lung cancer stem cells. To discover the pharmacological mechanism of AE on cancer stem cells will be helpful to develop new strategy for lung cancer therapy.

Note: This abstract was not presented at the meeting.

Citation Format: Pei-Jung Lee, Wan-Jiun Chen, Chao-Chi Ho, Pan-Chyr Yang, Huei-Wen Chen. Aloe-emodin as a potential lead targets lung cancer stem 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 133. doi:10.1158/1538-7445.AM2017-133
Abstract 1333: Cross talk between mitogen-activated protein kinase signaling cascades and Wnt/β-catenin in HER2+ overexpressing breast cancer cells
Authors: Nava Miguel , Amobi Nwamaka A. , Wu Yanyuan , Farias-Eisner Robin , Vadgama Jay
Published: July 2017

Introduction: The HER2-positive breast cancer is characterized by an amplification of the HER2 gene, resulting in an increase in HER2 presence on the surface of cells and magnification of downstream intracellular signaling. Treatment of HER2+ breast cancer with trastuzumab decreases tumor volume, reduces metastasis and increases overall survival. Unfortunately, HER2-positive breast cancers develop resistance to trastuzumab. The mechanism of resistance may involve the activation of compensatory networks of different cell signaling pathways. The purpose of the study is to examine the crosstalk between HER2/EGFR and Wnt signaling in HER2-positive breast cancer cells.

Methods: In order to address this, a HER2-positive breast cancer cell line (SKBR3) was used and induced with EGF. The phosphorylation of p38 and ERK1/2 was determined by Western blot analysis. The cellular localization of proteins of interest was examined using biochemically fractionated lysates followed by western blot analysis.

Results: Our data demonstrated an early and transient peak of phospho-p38 at 30min post EGF treatment. Interestingly, both phospho-p38 and phospo-ERK1/2 were detected in the nuclear compartment at this time. β-catenin, the major effector molecule in Wnt signaling, was stabilized 30min post EGF treatment. β-catenin protein levels remained higher in EGF treated cells than untreated cells up to 4h post EGF treatment. It is likely that a stabilized β-catenin following EGF treatment potently modulates a subset of Wnt targets. We are assessing this possibility using chromatin immunoprecipitation based experiments for H3K18ac, H3K27ac, pol2 and β-catenin. Taken together, our data suggests that EGFR signaling promotes Wnt signaling in HER2-positive breast cancer cells. We hypothesize that this promotes cancer stem cell niche maintenance and an EMT phenotype.

Citation Format: Miguel Nava, Nwamaka A. Amobi, Yanyuan Wu, Robin Farias-Eisner, Jay Vadgama. Cross talk between mitogen-activated protein kinase signaling cascades and Wnt/β-catenin in HER2+ overexpressing breast 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 1333. doi:10.1158/1538-7445.AM2017-1333
Abstract 1352: The importance of the RASA1/R-Ras/Ral-A signaling axis in melanoma tumorigenesis
Authors: Hill Kristen Suzanne , Wang Xue , Kim Youngchul , Kim Minjung
Published: July 2017

The Ras family of small GTP binding proteins are frequently activated by mutations in melanoma, as shown for NRAS (20%), KRAS (2%) and HRAS (1%). Ras isoforms can also be activated by inactivation of Ras GTPase activating proteins (RasGAPs), such as NF1, RASA1, and RASA2. In our recent study, we observed that inactivation of RASA1 (RAS p21 protein activator 1, also called p120RasGAP) suppressed melanoma via its RasGAP activity toward the R-Ras (related RAS viral (r-ras) oncogene homolog) isoform and that R-Ras was required to promote anchorage-independent growth driven by RASA1 inactivation. Moreover, a low level of RASA1 mRNA expression is associated with decreased overall survival in melanoma patients with BRAF mutations. Based on these observations, we hypothesized that, although not mutated, R-Ras is activated in melanoma by inactivation of RasGAPs and that BRAF activation cooperates with this RasGAP/R-Ras pathway activation in melanoma tumorigenesis. In this study, we addressed the importance of R-Ras, a previously less appreciated member of the Ras small GTPases family, in melanoma tumorigenesis. We observed frequent activation of R-Ras in BRAF mutant human melanoma cell lines. In addition, RNAi-mediated reduced expression of R-Ras suppressed anchorage-independent colony growth and tumor growth. Moreover, among the 3 major RAS effector pathways, reduced R-Ras expression suppressed Ral-A activation, which may explain the mechanism of Ral-A activation in BRAF mutant melanoma. Interestingly, anchorage-independent growth driven by R-Ras activation downstream of RASA1 inactivation was suppressed by both genetic (siRNA targeting Ral-A) and pharmacological (Ral inhibitor BQU57) inhibition of Ral-A. To further investigate the impact of RASA1 loss, and thus R-Ras activation, on BRAF mutant melanoma development in vivo, we generated a RASA1L/L; BRAFCA/CA; Tyr-CreERT2 mouse model in which treatment with 4OHT results in expression of constitutively activated mutant BRAF and deletion of RASA1 in melanocytic lineage cells. Preliminary analysis shows hyperpigmentation of the ear, tail, and foot pad in RASA1L/L BRAFCA/CA mice compared to RASA1+/+ BRAFCA/CA littermates. Tumors generated in this animal model will be analyzed to determine the extent of R-Ras and Ral-A activity in vivo. This study demonstrates the importance of the RASA1/R-Ras/Ral-A signaling pathway in BRAF mutant melanoma and supports the possible combinatorial treatment strategy targeting both the BRAF/MAPK and Ral signaling pathways.

Citation Format: Kristen Suzanne Hill, Xue Wang, Youngchul Kim, Minjung Kim. The importance of the RASA1/R-Ras/Ral-A signaling axis in melanoma 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 1352. doi:10.1158/1538-7445.AM2017-1352
Abstract 1367: The dynamic mechanism of SARAH domain in RASSF5 activation by K-Ras4B
Authors: Liao Tsung-Jen , Jang Hyunbum , Tsai Chung-Jung , Fushman David , Nussinov Ruth
Published: July 2017

RASSF5 is a tumor suppressor, which acts as an adaptor linking Ras and the Hippo pathways. RASSF5 activates MST1/2, a key upstream protein of the Hippo pathway, through SARAH domain heterodimerization, resulting in Hippo signaling. Signaling stimulates YAP1 phosphorylation, leading to its degradation and thereby cell apoptosis. Association with K-Ras4B promotes RASSF5 to release its SARAH which heterodimerizes with MST1/2. We aim to reveal how K-Ras4B regulates the Hippo pathway through RASSF5 by resolving the structure and conformational dynamics of RASSF5 complexes. Our modeling and simulations help elucidate the intrinsic dynamic interactions of RASSF5 RA (Ras binding) and SARAH domains. Surprisingly, we found that the residue E388 on RASSF5 SARAH plays a significant role in the dynamic mechanism of SARAH homo-/hetero- dimerization and the RA-SARAH association. The mutants E388A and E388K dramatically decrease the strength of RASSF5 homo-SARAH and RASSF5-MST2 hetero-SARAH, suggesting that the E388 mutation may be oncogenic. Simulations of selected RASSF5 configurations with GTP-bound K-Ras4B indicate that the interactions between RA and SARAH are reduced. We conclude that Ras binding weakens the RA-SARAH association, increasing the SARAH dynamic fluctuation. The released RASSF5 SARAH has high propensity to dimerize with MST1/2 SARAH, activating Hippo signaling thus cancer suppression. Funded in whole or in part with Federal funds from the Frederick National Laboratory for Cancer Research, NIH under contract HHSN261200800001E.

Citation Format: Tsung-Jen Liao, Hyunbum Jang, Chung-Jung Tsai, David Fushman, Ruth Nussinov. The dynamic mechanism of SARAH domain in RASSF5 activation by K-Ras4B [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 1367. doi:10.1158/1538-7445.AM2017-1367
Abstract 1370: EGFR-mediated Spred1 phosphorylation inhibits NF1 to sustain constitutive Ras/MAPK signaling
Authors: Markegard Evan , Mercado Ellen L. , Silva Jillian M. , Galeas Jacqueline , Trinidad Marena I. , Urisman Anatoly , McCormick Frank
Published: July 2017

Spred proteins negatively regulate Ras/MAPK signaling following growth factor stimulation. Inhibition of Ras primary occurs through Spreds ability to bind and localize NF1, a RasGAP and major tumor suppressor, to the plasma membrane. Spred1 and NF1 loss-of-function mutations occur across multiple cancer types including non-small cell lung carcinoma, glioblastoma, melanoma, stomach carcinoma, and uterine carcinosarcoma. Here we demonstrate that EGFR signaling disrupts Spred1-NF1 binding. Mass spectrometry was performed on cells overexpressing EGFRL858R to identify potential phosphorylation sites on Spred1 and NF1 that could disrupt Spred1-NF1 binding by steric hindrance. A serine phosphorylation site on Spred1 was identified in which a phosphomimetic and phosphodeficient mutant decreased or increased Spred1-NF1 binding, respectively. Phosphomimetic Spred1 is unable to suppress Ras-GTP following EGF stimulation. Therefore, phosphorylation of Spred1 at this site by a serine kinase downstream of EGFR may disrupt Spred1-NF1 binding. To identify the Spred1 kinase we are performing an in vitro kinase assay and an unbiased CRISPRa screen. Our findings provide one potential mechanism by which EGFR signaling disrupts negative feedback to sustain constitutive Ras signaling. Furthermore, this work may elucidate a novel therapeutic target for restoring NF1-mediated inhibition of Ras.

Citation Format: Evan Markegard, Ellen L. Mercado, Jillian M. Silva, Jacqueline Galeas, Marena I. Trinidad, Anatoly Urisman, Frank McCormick. EGFR-mediated Spred1 phosphorylation inhibits NF1 to sustain constitutive Ras/MAPK signaling [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 1370. doi:10.1158/1538-7445.AM2017-1370
Abstract 1380: Expression and functions of ASH1L in liver cancer
Authors: Wei Lai , Tsang Felice H. , Ho Daniel , Law Cheuk-Ting , Chen Mengnuo , Tsang Long-Hin , Wong Carmen Cl , Ng Irene Ol , Wong Chun-Ming
Published: July 2017

Hepatocellular carcinoma (HCC) is a common cancer and ranks the third lethal cancer worldwide. However, the detail molecular mechanisms underlying the initiation and progression of HCC remain poorly understood. Liver carcinogenesis is a multistep process that is driven by the accumulation of genetic and epigenetic alterations. Mutational landscape and driver mutations have recently been delineated by high-throughput sequencing studies. On the other hand, our current knowledge about epigenetic deregulation in human HCC is limited. Histone modification is a major component of epigenetic regulation, and deregulation of histone medication could alter local chromatin structure and gene expression. We hypothesized that deregulation of histone methyltransferases might contribute to HCC development. In this study, we showed that trithorax-group protein ASH1L was frequently up-regulated in human HCC. Overexpression of ASH1L was detected in 40% of primary HCCs and significantly associated with larger tumor size, present of venous invasion and tumor microsatellite formation. Up-regulation of ASH1L also correlated with increased Ki67 expression. ASH1L is a histone methyltransferase specific for catalyzing transcriptional-active H3K4 and H3K36 methylation. We showed that stable knockdown of ASH1L significantly suppressed HCC cell proliferation and colony formation. We further demonstrated that knockdown of ASH1L inhibited HCC cell migration. In addition, we investigated the underlying mechanisms contribute to ASH1L up-regulation. We showed that up-regulation of ASH1L in human HCC was contributed by chromosome amplification at chromosome 1q22. Furthermore, we showed that ASH1L was negatively regulated by miR-142. Therefore, down-regulation of miR-142 contributed to ASH1L up-regulation in HCC. In summary, our findings suggested that ASH1L was frequently up-regulated in human HCC due to chromosome amplification and miRNA deregulation.

Citation Format: Lai Wei, Felice H. Tsang, Daniel Ho, Cheuk-Ting Law, Mengnuo Chen, Long-Hin Tsang, Carmen Cl Wong, Irene Ol Ng, Chun-Ming Wong. Expression and functions of ASH1L in liver 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 1380. doi:10.1158/1538-7445.AM2017-1380
Abstract 1388: SphK2/S1P axis regulates hypoxia-mediated HIF-α synthesis in breast cancer cells
Authors: Hait Nitai C. , Maiti Aparna
Published: July 2017

The hypoxia-inducible transcription factors (HIF)-1α and -2α play a critical role in cellular response to hypoxia in solid tumors. Elevated HIF-α expression correlates with poor patient survival in a large number of cancers including breast cancers. HIF-α activates expression of genes promoting angiogenesis, metastasis, increased tumor growth and resistance to treatments. Understanding transcriptional regulation HIF-α under physiologically relevant hypoxic conditions would be helpful to reduce breast cancer growth. Our recent studies identified that intracellular bioactive sphingosine-1-phosphate (S1P) generated from nuclear sphingosine kinase 2 (SphK2) acts as an endogenous modulator of histone deacetylases (HDACs) epigenetically regulates gene transcription in breast cancer cells. We wonder whether nuclear S1P epigenetically regulates acetylated histones, positive transcription marks in hypoxia and regulate transcription of HIF-α and target genes in breast cancer cells. We have used estrogen receptor positive human breast cancer MCF-7 cells, triple-negative human breast cancer MDA-231 cells, and lung metastatic LM-2-4 cells derived from MDA-231 cells cells for our studies. We found that hypoxic condition enhances nuclear bulk histone acetylation in breast cancer cells and enhances HIF-α synthesis. Down regulation of SphK2 with siRNA or using selective inhibitor of SphK2 (K145 compound) reduces nuclear S1P as well as bulk histone acetylation and consequently reduces nuclear HIF-α synthesis in breast cancer cells. Downregulation of cytosolic sphingosine kinase 1 (SphK1) with siRNA reduces cytosolic S1P does not alter nuclear bulk histone acetylation neither significantly alter nuclear HIF-α synthesis in breast cancer cells. We also found that targeting SphK2 with K145 compound dramatically reduces in vitro tumorosphere formation in breast cancer cells. Our data suggested that targeting the SphK2/S1P signaling could represent an attractive strategy for therapeutic intervention in hypoxic breast cancers. Supported by Health Research, Inc. (HRI) Grant 71-4084 (NCH), National Cancer Institute Grant R01CA160688 (KT).

Citation Format: Nitai C. Hait, Aparna Maiti. SphK2/S1P axis regulates hypoxia-mediated HIF-α synthesis in breast 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 1388. doi:10.1158/1538-7445.AM2017-1388
Abstract 1391: The epigenetic regulator, G9a, is a KRAS-inducible protein and its inactivation inhibits PanIN formation by this oncogene
Authors: Mathison Angela , Salmonson Ann , Paradise Brooke , Missfeldt Mckenna , Iovanna Juan , Billadeau Daniel , Urrutia Raul , Lomberk Gwen
Published: July 2017

Pancreatic ductular adenocarcinoma (PDAC) ranks fourth as a cause of cancer death in the USA and is almost universally fatal, with the annual number of deaths equivalent to the number of newly diagnosed cases. Valuable research in the field has revealed genetic aberrations that contribute to PDAC development and progression, with KRAS being one of the most frequent mutations in more than 90% of patient samples. However, to date, any efforts to directly target KRAS have failed in the clinic. Thus, there is indisputably an urgent need to further improve our understanding of molecular mechanisms underlying PDAC development as to identify novel therapeutic targets, including druggable important downstream targets and nodes orchestrated by oncogenic KRAS. In particular, we are interested in epigenetic pathways involved in PDAC development and progression due to the potential reversibility of any alteration, unlike genetic mutation. In the current study, using a cell model that allows inducible expression of mutant KRASG12D, we find that protein levels of the dimethyl-K9H3 histone methyl transferase (HMT), G9a, and its complex partners are increased in response to activation of the oncogenic Kras pathway. Furthermore, the activation of this oncogenic pathway results in the formation of the G9a-GLP-Wiz trimer complex, as determined by affinity protein purification, combined with mass spectrometry. In vivo experiments involving the cross of the Pdx1-CRE/LSL-KRASG12D mice with G9afl/fl animals demonstrate that a loss of the H3K9Me2 mark in the nucleus of exocrine cells is accompanied by a significantly reduced number of PanIN lesions. RNA-Seq experiments from these animals reveal that these mice have reduced levels of typical molecular markers of PanINs. In addition, these experiments show changes in the levels in several genes, which have been previously been shown to synergize with Kras to mediate pancreatic cancer initiation. Congruently, pharmacological inhibition of G9a using BRD4770 displays an inhibitory effect on KRASG12D-induced cell proliferation. Combined, these data provide evidence for a key role of the meK9H3-G9a pathway as a mediator of the oncogenic Kras response and defines a novel point of potential therapeutic intervention for PDAC.

Citation Format: Angela Mathison, Ann Salmonson, Brooke Paradise, Mckenna Missfeldt, Juan Iovanna, Daniel Billadeau, Raul Urrutia, Gwen Lomberk. The epigenetic regulator, G9a, is a KRAS-inducible protein and its inactivation inhibits PanIN formation by this oncogene [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 1391. doi:10.1158/1538-7445.AM2017-1391
Abstract 1394: Bioorthogonal Profiling of Protein Methylation (BPPM) identified MCM5 as a new substrate for SETD8 in DNA replication
Authors: Silva Fabio Pittella , Blum Gil , Senevirathne Chamara , Minkui Luo
Published: July 2017

SETD8 is the only member of the SET domain containing methyltransferase family, which catalyzes mono-methylation of K20 on histone H4 (H4K20me1). Lysine residues of non-histone proteins such as p53 and proliferating cell nuclear antigen (PCNA) are also monomethylated, while lysine residues in Numb were found to be dimethylated. As a consequence, SETD8 methyltransferase activity is implicated in several fundamental cellular processes such as transcriptional regulation and heterochromatin formation as well as processes that ensures genomic stability including DNA replication and the DNA damage response. Although it has been suggested that SETD8 is involved in DNA replication as a positive regulator of origin licensing through H4K20 methyation and by supporting Okazaki fragment processing through PCNA methylation, to date, there is no evidence whether other key protein in the replication fork is directly modified by SETD8. To address this question, we used Bioorthogonal Profiling of Protein Methylation (BPPM) with engineered enzyme and synthetic SAM analogues to profile new substrates for SETD8. We genetically engineered SETD8 and identified mutants amenable to accommodate non-native SAM analogues containing a terminal alkyne moiety for click chemistry. The engineered SETD8 can transfer this distinct chemical moiety into target proteins for subsequent pulldown and identification of the modified substrates. Among the new substrates discovered, we identified MCM5, a subunit of the hexameric minichromosome maintenance (MCM) DNA helicase complex. MCM5 directly interacts with MCM2 and 3 to form the MCM2-7 hexamer which associates with the origins of DNA replication to form part of the pre-replicative complex (preRC), playing a key role during replication initiation and elongation. We found that SETD8 can mono-methylate MCM5 directly affecting its binding affinity to MCM2 and 3. In addition, MCM5 mutations at the methylated lysine further evidenced a stronger binding to its interacting partners contributing to the formation of the MCM hexamer. Taken together our results indicate that MCM5 methylation contributes to the assembly of the MCM complex suggesting an important role for SETD8 in replication initiation. Our findings may bring new perspectives on the biological importance of SETD8 during DNA replication.

Citation Format: Fabio Pittella Silva, Gil Blum, Chamara Senevirathne, Luo Minkui. Bioorthogonal Profiling of Protein Methylation (BPPM) identified MCM5 as a new substrate for SETD8 in DNA replication [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 1394. doi:10.1158/1538-7445.AM2017-1394
Abstract 1398: Loss of PIWIL4 and piRNAs attenuate somatic methylome and genome stability
Authors: Lin Syuan-Cih , Chiu Chia-Chen , Chen Shou-Tung , Chen Yao-Li , Lin Ping-Yi , Hsiao Shu-Huei , Leu Yu-Wei
Published: July 2017

Piwi-like RNA-mediated gene silencing 4 (PIWIL4) is expressed in somatic tissues and is proposed to silence the transposition of repetitive elements like long interspersed nuclear elements (LINEs) through DNA methylation and maintain the genome stability. Through the knockout of PIWIL4 gene by CRISPR/Cas9 system in mesenchymal stem cells (MSCs), MDA-MB-231 (breast cancer cell line), HT29 (colon cancer cell line) and AGS (gastric cancer cell line), the piRNA biogenesis was decreased and so was the methylation within LINE1 target loci and global methylation. The recruitment of SUV39H1, a histone H3 methyl-transferase, into nucleus was blocked, leading to the reduction of silencing tri-methylation of histone H3 at lysine 9. Concurrently, the active H3K4me3 mark increased after knockout. Also, global CTCF bindings were distorted after the knockout of PIWIL4 especially at the bindings franking the piRNA clusters. This distortion implied the disorganization of genomic conformation which was validated by karyotyping. Through chromosomal G-banding, aneuploidy was observed which indicated the loss of genomic stability. The knockout of PIWIL4 also results in disorganized cytoskeleton, therefore, changed cell stiffness as measured by atomic force microscopy. Neural and hepatic induction of MSCs were also blocked by the knockout of PIWIL4. In correlation, tissue arrays detected abnormal PIWIL4 expression in gastric (n=45), colon (n=30) and breast cancers (n=30). In conclusion, we found that PIWIL4-piRNA interaction helps to maintain somatic DNA methylome, genome stability and stiffness in somatic stem cells. The loss of PIWIL4 expression might then a candidate for cancer biomarker. (Supported by: MOST-105-2314-B-371 -008, MOST-105-2320-B-194 -004, MOST Taiwan)

Citation Format: Syuan-Cih Lin, Chia-Chen Chiu, Shou-Tung Chen, Yao-Li Chen, Ping-Yi Lin, Shu-Huei Hsiao, Yu-Wei Leu. Loss of PIWIL4 and piRNAs attenuate somatic methylome and genome stability [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 1398. doi:10.1158/1538-7445.AM2017-1398
Abstract 1399: In vitro characterization of several enzymes of the Fe(II)/2-oxoglutarate-dependent dioxygenase superfamily using a universal succinate detection assay
Authors: Alves Juliano , Goueli Said , Zegzouti Hicham
Published: July 2017

The covalent modification of histone proteins, DNA and RNA by Fe(II)/2-oxoglutarate-dependent dioxygenases are key to the modulation of biological processes such as epigenetics, hypoxic signaling and DNA/RNA repair. Of these, JumonjiC domain-containing histone lysine demethylases (JMJCs), the ten-eleven-translocation (TET) DNA dioxygenases, the ALKB DNA/RNA hydroxylases and the prolyl hydroxylases EGLN1-3 have generated increased interest as potential drug targets for the treatment of a number of pathological conditions, including cancer. Since succinate is a common product to all Fe(II)/2-oxoglutarate-dependent dioxygenases, we determined the substrate specificities, the apparent kinetic constants for several JMJCs and members of the dioxygenase superfamily (i.e.: TET1, EGLN1, ALKBH3 and FTO), as well as inhibition profiles of reported inhibitors using a novel bioluminescent and homogenous succinate detection assay. Our results demonstrate that succinate detection is a useful strategy for the characterization of multiple Fe(II)/2-oxoglutarate-dependent dioxygenases with distinct substrate requirements, enabling the investigation of a large number of enzymes that cannot be evaluated in a miniaturized or high-throughput manner with the methods currently available.

Citation Format: Juliano Alves, Said Goueli, Hicham Zegzouti. In vitro characterization of several enzymes of the Fe(II)/2-oxoglutarate-dependent dioxygenase superfamily using a universal succinate detection assay [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 1399. doi:10.1158/1538-7445.AM2017-1399
Abstract 1400: Using tumor spheroids to evaluate the efficacy of EZH2 inhibitors in clear cell renal cell carcinoma (ccRCC)
Authors: Wei Darmood , Yang Youfeng , Ricketts Christopher J. , Sourbier Carole , Schmidt Laura S. , Linehan William M.
Published: July 2017

Enhancer of zeste homolog 2 (EZH2) is a key component of the polycomb repressive complex 2 (PRC2). EZH2 is frequently overexpressed in a wide variety of human malignancies including non-Hodgkin lymphoma, gastric cancer, pancreatic cancer, and lung cancer. Thus it has potential to become a therapeutic target. Characterization of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC) has not been fully explored. ccRCC have been defined by mutation of the von Hippel-Lindau (VHL) tumor suppressor gene in combination with chromosome 3p loss. Recent sequencing efforts have revealed that several chromatin remodeling genes encoded on chromosome 3p are often mutated, of which PBRM1 is the most frequent (41%). The PBRM1 gene codes for the BAF180 protein, a SWI/SNF chromatin remodeling complex subunit. Loss of BAF180 in ccRCC may disrupt the PBAF variant of the SWI/SNF complex. The SWI/SNF complex remodels the chromatin landscape by either sliding or evicting the nucleosomes from the chromatin. This chromatin remodeling modulates the accessibility to promoter regions by transcriptional machinery. It is through this mechanism that the SWI/SNF complex can regulate a range of cellular processes. It has been demonstrated that the SWI/SNF complex can act antagonistically to the PRC2 complex by evicting PRC2 complex from the promoters of tumor suppressors. Disruption of the SWI/SNF complex would impede the eviction of the PRC2 complex, similarly observed in SNF5-deficient malignant rhabdoid tumors. Therefore, we hypothesize that PBRM1 inactivation disrupts specific SWI/SNF complexes allowing EZH2 to bind and repress target tumor suppressor genes. Thus inhibition of EZH2 in ccRCC may present as a targeted therapeutic option in tumors with PBRM1 mutations. We have investigated EZH2 in ccRCC cell lines with PBRM1 mutations and observed that these cells lines have overexpression of EZH2 in comparison to RPTEC (renal cortex proximal tubule epithelium cell line). We examined the effects of two EZH2 inhibitors (GSK126 and EPZ6438) on ccRCC tumor spheroids. Our preliminary data suggests EZH2 inhibition results in reduced growth of PBRM1 mutant cell lines grown as tumor spheroids.

Citation Format: Darmood Wei, Youfeng Yang, Christopher J. Ricketts, Carole Sourbier, Laura S. Schmidt, William M. Linehan. Using tumor spheroids to evaluate the efficacy of EZH2 inhibitors in clear cell renal cell carcinoma (ccRCC) [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 1400. doi:10.1158/1538-7445.AM2017-1400
Abstract 1402: NME3 links mitochondrial fusion to DNA repair in nuclear genome
Authors: Chang Zee-Fen
Published: July 2017

In mammals, there are 10 isoforms of NME (NDP Kinase), which catalyze the reversible reaction of NTPs formation from NDP and ATP. By RNA interference, NME3 was found critical for DNA repair. Data from analysis of the steady-state levels of rNTPs and dNTPs suggest that NME3 does not play a critical role in determining the total pools of these nucleotides. However, its mitochondrial localization and catalytic function are necessary for its function in DNA repair. Super-resolution microscopy analysis indicates the localization of NME3 at mitochondrial outer membrane. We further used APEX-mediated biotinylation to identify proteins interacting with NME3. Among them, Mfn1 is validated to associate with mitochondrial NME3 in vitro and in vivo. We provide evidence that NME3 is required for DNA damage-induced mitochondria fusion via GTP-mediated fueling of Mfn1. The lack of this process increased nucleotide oxidation and sustained DNA damage in nuclear genome. These data highlight the function of NME3 in mitochondrial fusion, which is required for the maintenance of genome integrity.

Citation Format: Zee-Fen Chang. NME3 links mitochondrial fusion to DNA repair in nuclear genome [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 1402. doi:10.1158/1538-7445.AM2017-1402
Abstract 1410: Proliferation during epithelial-to-mesenchymal transition induces genomic instability
Authors: Comaills Valentine , Kabeche Lilian , Morris Robert , Yu Min , Madden Marissa Wells , LiCausi Joseph A. , Aceto Nicola , Zheng Yu , Miyamoto David T. , Ramaswamy Sridhar , Zou Lee , Haber Daniel A. , Maheswaran Shyamala
Published: July 2017

Epithelial to mesenchymal transition (EMT), a morphogenetic process required for proper embryonic development, is adopted by cancer cells during tumor progression. We show that induction of EMT by TGFß or other EMT-inducers such as Snail leads to genomic instability, associated with failed cytokinesis and chromosome missegregation resulting in aneuploidy and polyploidy. These defects are dependent on persistent proliferation of cells undergoing EMT and are absent in normal cells that growth arrest during EMT. While EMT and the associated mitotic abnormalities are reversible upon removal of the EMT-inducer, the resulting chromosomal abnormalities are inherited. TGFß-induced genomic instability is associated with the acquisition of tumorigenic phenotypes and the resulting tumors are enriched for a genomically-altered tumor cell population, which exhibits differential drug sensitivity. In breast and prostate cancer cells circulating in the blood, TGFß and EMT gene signatures are significantly correlated with aneuploid gene signatures. Analysis of single circulating tumor cells (CTC) from women with metastatic breast cancer reveals increased in genomic instability in mesenchymal CTCs. Together, these findings identify a novel mechanism whereby tumor microenvironment-derived signals impact heritable genetic changes within cancer cells.

Citation Format: Valentine Comaills, Lilian Kabeche, Robert Morris, Min Yu, Marissa Wells Madden, Joseph A. LiCausi, Nicola Aceto, Yu Zheng, David T. Miyamoto, Sridhar Ramaswamy, Lee Zou, Daniel A. Haber, Shyamala Maheswaran. Proliferation during epithelial-to-mesenchymal transition induces genomic instability [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 1410. doi:10.1158/1538-7445.AM2017-1410