bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1: Noxa and ceramide: Crosstalk between Bcl-2 family members and ceramide in mediating intrinsic apoptosis in Molt-4 human T-cell leukemia
Authors: Kobeissy Hadile , Hage-Sleiman Rouba , Dbaibo Ghassan
Published: August 2015

The sphingolipid breakdown product, ceramide, has been proposed as a coordinator of many cellular processes, one of which is apoptosis. We have previously shown that γ-irradiation induces p53-dependent apoptosis through ceramide accumulation in Molt-4 human T-cell leukemia cell line. Noxa, a BH3-only pro-apoptotic protein and a member in the Bcl-2 family, is activated by p53 and regulates downstream proteins that initiate mitochondrial outer membrane permeabilization (MOMP) and subsequently apoptosis. Here we identify a novel link between Noxa and ceramide in the apoptotic pathway that were previously thought to work independently. Specifically, we knocked down either p53 or Noxa protein expression in γ-irradiated Molt-4 cells and measured ceramide level in response to γ-irradiation. Our results demonstrate that ceramide accumulation requires the expression of both p53 and Noxa whereas inhibition of either p53 or Noxa totally inhibits ceramide accumulation. Furthermore, lipid and protein analysis of mitochondria isolated from Molt-4 cells showed a simultaneous ceramide accumulation and cytochrome c release, suggesting that ceramide's role is intricately related to the execution phase of apoptosis. These findings establish a role for Noxa in regulating ceramide at the mitochondrial level and provide a better understanding of the mechanisms of apoptosis initiated by p53.

Citation Format: Hadile Kobeissy, Rouba Hage-Sleiman, Ghassan Dbaibo. Noxa and ceramide: Crosstalk between Bcl-2 family members and ceramide in mediating intrinsic apoptosis in Molt-4 human T-cell leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1. doi:10.1158/1538-7445.AM2015-1
Abstract 1015: Misaponin B induces G2-M arrest and autophagy via upregulation of miR1290 in non-small cell lung cancer (NSCLC) A549 cells
Authors: Won Gunho , Sohn Eun Jung , Jeong Myoung Seok , Yoon Sang Wook , Lee Jihyun , Kim Bonglee , Kim Sung Hoon
Published: August 2015

Though Mi-saponin B is found to be the potent component among a mixture of saponins, its function still remains unlcear. Thus, in the present study, we investigated the anti-tumor mechanism of Mi-saponin B in non-small cell lung cancer (NSCLC) A549 cells. Mi-saponin B treatment caused cell growth inhibition via G2/M arrest in A549 cells. Moreover, Western blotting and qRT-PCR assays showed that Mi-saponin B induced conversion of LC3I to LC3II, a maker of autophagy. Immunofluorescence assay revealed that Mi-saponin B treatment formed the puncta of GFP-fused LC3II. Conversely, 3MA, an inhibitor of autophagy, attenuated the LC3II expression induced by Mi-saponin B in A549 cells. Also, transmission electron microscopy (TEM) showed that Mi-saponin B induces the autophagic vacuoles. Interestingly, miR1290 was upregulated in a dose dependent manner in Mi-saponin B treated A549 cells by qRT-PCR. Also, combined treatment of miR1290 and Mi-saponin B increased the expression of LC3II at protein and mRNA levels and also enhanced the formation of puncta of LC3II in A549 cells. Overall, our findings for the first time demonstrate that Mi-saponin B induces G2/M arrest and autophagy via upregulation of miR1290 in A549 cells as a potent anti-cancer agent.

Citation Format: Gunho Won, Eun Jung Sohn, Myoung Seok Jeong, Sang Wook Yoon, Jihyun Lee, Bonglee Kim, Sung Hoon Kim. Misaponin B induces G2-M arrest and autophagy via upregulation of miR1290 in non-small cell lung cancer (NSCLC) A549 cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1015. doi:10.1158/1538-7445.AM2015-1015
Abstract 1022: DYRK1A kinase regulates mTOR signaling via modulating the TSC complex
Authors: Menon Vijay R. , Litovchick Larisa
Published: August 2015

The Dual specificity Tyrosine-phosphorylation-Regulated Kinase 1A (DYRK1A) kinase is encoded on chromosome 21 and is involved in the pathogenesis of Down syndrome. Earlier studies have shown that DYRK1A is essential for cells to undergo growth arrest following serum starvation, at least in part by promoting the assembly of the DREAM repressor complex. However, the function of this important kinase is not fully understood. Using MudPIT MS/MS proteomic analysis of DYRK1A-interacting proteins followed by immunoprecipitation-Western blotting, we characterized the binding between DYRK1A and the subunits of the TSC tumor suppressor protein complex, a major regulator of the mTORC1 pathway. This interaction was mapped to the kinase domain of DYRK1A, suggesting that the TSC could serve as a substrate for DYRK1A phosphorylation. Indeed, overexpression of DYRK1A increased phosphorylation of both TSC1 and TSC2; this effect was fully reversed by the DYRK1A inhibitor, harmine. Functional studies revealed that loss of DYRK1A in two different cancer cell lines resulted in increased phosphorylation of mTORC1 substrates S6K1 and 4E-BP1. This effect of DYRK1A knockdown was inhibited by rapamycin, confirming the involvement of mTORC1 signaling. Thus, our work revealed novel function of DYRK1A in the regulation of mTOR signaling that controls cell growth, proliferation, and survival.

Citation Format: Vijay R. Menon, Larisa Litovchick. DYRK1A kinase regulates mTOR signaling via modulating the TSC complex. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1022. doi:10.1158/1538-7445.AM2015-1022
Abstract 1025: Protein kinase D2 promotes the proliferation of glioma cells by regulating golgi phosphoprotein 3
Authors: Zhou Xiuping , Xue Pengfei , Yang Minglin , Shi Hengliang , Lu Dong , Shi Qiong , Hu Jinxia , Yu Rutong
Published: August 2015

Protein kinase D2 (PKD2) has been demonstrated to promote tumorigenesis in many types of cancers. However, how PKD2 regulates cancer cell growth is largely unknown. In this study, we found that over-expression of PKD2 promoted glioma cell growth but down-regulation of PKD2 inhibited it. Further investigation indicated that PKD2 down-regulation decreased the protein level of Golgi phosphoprotein 3(GOLPH3) as well as p-AKT level. On the contrary, over-expression of PKD2 increased the protein level of GOLPH3 and p-AKT. In addition, GOLPH3 exhibited similar effect on glioma cell growth to that of PKD2. Interestingly, GOLPH3 down-regulation partially abolished glioma cell proliferation induced by PKD2 over-expression, while over-expression of GOLPH3 also partially rescued the inhibition effect of PKD2 down-regulation on glioma cell growth. Importantly, the level of PKD2 and GOLPH3 significantly increased and was positively correlated in a cohort of glioma patients, as well as in patients from TCGA database. Taken together, these results reveal that PKD2 promotes glioma cell proliferation by regulating GOLPH3 and then AKT activation. Our findings indicate that both PKD2 and GOLPH3 play important roles in the progression of human gliomas and PKD2-GOLPH3-AKT signaling pathway might be a potential glioma therapeutic target.

Citation Format: Xiuping Zhou, Pengfei Xue, Minglin Yang, Hengliang Shi, Dong Lu, Qiong Shi, Jinxia Hu, Rutong Yu. Protein kinase D2 promotes the proliferation of glioma cells by regulating golgi phosphoprotein 3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1025. doi:10.1158/1538-7445.AM2015-1025
Abstract 1033: Alternative tumor suppressor mechanisms in lung carcinomas - modulation of senescence by growth factor-induced signaling
Authors: Markova Boyka , Breitenbuecher Frank , Schuler Martin
Published: August 2015

Cellular signaling pathways integrate multiple intra- and extracellular signals and thereby influence cell fate. Outcome of oncogene-targeted pharmacotherapies (cell cycle inhibition, apoptosis, senescence, autophagy) depends on variable factors (e.g. compound, dose, duration, cellular genetic background etc.). Oncogene-induced senescence is recognized as an important tumor-suppressive mechanism, bypassing of which may contribute to malignant transformation of the cells. On the other hand, senescence induction, in addition to apoptosis stimulation and proliferation suppression, is responsible for the cytostatic activity of many commonly used anticancer drugs. Therefore, senescence escape must be considered as an important drug-resistance factor.

In our work we characterize,,DNA Damage-independent” senescence induced by tyrosine kinase inhibitors (TKI) and growth factor deprivation of non-small cell lung cancer (NSCLC) cell lines and other EGFR-dependent cells.

Treatment of different cell lines with various concentrations of EGFR-TKIs was followed by the assessment of cell fate (i.e. apoptosis, cell cycle distribution, senescence and clonogenic survival) as well as intracellular signaling. We could show that treatment of EGFR-dependent cells with specific EGF receptor tyrosine kinase inhibitors (EGFR-TKI) can lead to generation of reactive oxygen species (ROS), activation of a DNA damage response (DDR) program and cellular senescence. Surprisingly, under certain experimental conditions DDR activation was found to be independent from detectable DNA damage. Key players of DDR signaling were shown to be essential for senescence induction.

Molecular dissection of crucial signaling nodes and molecular switches involved in modulation of cell fate in oncogene dependent cells treated with specific EGFR-inhibitors is currently being performed. Furthermore, our work is focussed on the identification and characterization of cellular factors activating DNA damage response (DDR) pathways in the absence of DNA damage under certain experimental conditions.

Understanding the interplay between theses decision making pathways either leading to proliferative arrest, cellular senescence or apoptosis will help improve current pharmacotherapies and develop new therapeutic approaches.

Note: This abstract was not presented at the meeting.

Citation Format: Boyka Markova, Frank Breitenbuecher, Martin Schuler. Alternative tumor suppressor mechanisms in lung carcinomas - modulation of senescence by growth factor-induced signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1033. doi:10.1158/1538-7445.AM2015-1033
Abstract 104: ATF7IP does not alter the substrate specificity of the lysine methyltransferase SETDB1
Authors: Basavapathruni Aravind , Gureasko Jodi , Porter Scott Margaret , Boriack-Sjodin P. Ann , Wigle Timothy J. , Riera Thomas V. , Copeland Robert A.
Published: August 2015

The histone methyltransferase (HMT) SETDB1 is a strong candidate oncogene in melanoma and lung carcinomas. SETDB1 methylates lysine 9 of histone 3 (H3K9), utilizing S-adenosylmethionine (SAM) as the methyl donor. Its activity has been shown to be regulated by its partner protein ATF7IP, and here we examine the contribution of ATF7IP to the in vitro activity and substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed in Sf21 insect cells and 1:1 stoichiometric complexes were purified for comparison against apo-SETDB1 enzyme. We employed both radiometric flashplate-based and MALDI mass spectrometry assays to follow methylation on histone 3 15-mer peptides where lysine 9 was either unmodified, mono-, or di-methylated. These two methods provide orthogonal readouts on H3K9 methylation: the radiometric flashplate-based assay employs 3H-labeled SAM and provides quantitation of labeled, methylated peptide. The MALDI mass spectrometry assay offers quantitation of consumption and accumulation of each individual H3K9 methylation state (H3K9me0, me1, me2, and me3). Results show that both apo-SETDB1 and SETDB1:ATF7IP complex catalyzed both monomethylation and dimethylation of H3K9 peptide substrates, but were unable to perform H3K9 trimethylation. While ATF7IP did not impact the substrate methylation profile, the activity of the complex was lower than apo-SETDB1 by a factor of 4. This difference was due to a decrease in the value of kcat as the substrate KM values were comparable between apo-SETDB1 and the SETDB1:ATF7IP complex. ATF7IP therefore does not alter SETDB1's substrate specificity. H3K9 monomethylation and dimethylation by SETDB1 occurred in a distributive manner and was unaffected by the presence of ATF7IP. This finding is important as H3K9 can be methylated by HMTs other than SETDB1 and a distributive mechanism would allow for interplay between multiple HMTs on H3K9me1 and H3K9me2. The results presented here indicate that ATF7IP does not alter substrate specificity of SETDB1 in vitro, and though it decreases SETDB1's catalytic activity, our data points to a nonenzymatic function of ATF7IP in regulating SETDB1 function.

Citation Format: Aravind Basavapathruni, Jodi Gureasko, Margaret Porter Scott, P. Ann Boriack-Sjodin, Timothy J. Wigle, Thomas V. Riera, Robert A. Copeland. ATF7IP does not alter the substrate specificity of the lysine methyltransferase SETDB1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 104. doi:10.1158/1538-7445.AM2015-104
Abstract 1057: Characterization of methylation patterns in cancer tissue shown by targeted Next-Gen bisulfite sequencing
Authors: Alexander Jessica , Drennan Ryan , Meyer Ann , Xu Jessica , Poulin Matthew L. , Timp Winston
Published: August 2015

Continuing research demonstrates an ever increasing number of genes with measurable dysregulation in their DNA methylation state.We have developed a panel of targeted NextGen bisulfite sequencing amplicon assays (TGBS) centered on cancer genes with altered DNA methylation. This cancer panel includes tumor suppressor genes (BRCA1, RASSF1, RUNX3), DNA methyltransferases (DNMTs and MGMT), DNA repeats (LINE1 and Sat2), oncogenes (MYC,ERBB2), receptor genes (EGFR, ESRRA, NR2E1), Homeobox genes (HOXA10, HOXA11, EN1) and detoxification genes (GSTM1, GSTM2) among others. A total of 80 genes covering over 1500 CpG sites were selected and assays for these regions were developed and validated using PCR/Pyrosequencing. 10 pairs of DNA samples (tumor vs adjacent normal) for four cancer types (ovarian cancer, breast cancer, cervical cancer, and colorectal cancer) were sequenced using this panel on an Ion Torrent PGM™ system. We have used this data to characterize the general behavior of these cancer samples as compared to normal in three specific ways. First, we have assayed the change in overall methylation level between normal and cancer samples using these multiplexed amplicons, using these values for both an unsupervised hierarchical clustering and principal component analyses. We have then attempted to establish a per tissue normal methylation profile, and measured the distance to this normal profile for each of the cancer samples. As has been discussed in previous work, cancer has more of an anti-normal profile than a characteristic pattern per se. Finally, we have examined the methylation patterns within individual reads, and assessed the measured methylation entropy or randomness of these patterns in normal versus cancer samples

Citation Format: Jessica Alexander, Ryan Drennan, Ann Meyer, Jessica Xu, Matthew L. Poulin, Winston Timp. Characterization of methylation patterns in cancer tissue shown by targeted Next-Gen bisulfite sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1057. doi:10.1158/1538-7445.AM2015-1057
Abstract 1070: Epigenetic silencing of galectin-12 in colorectal cancer
Authors: Katzenmaier Eva-Maria , Gebert Johannes , Kloor Matthias , Kopitz Jürgen
Published: August 2015

Aim/Background: Galectins constitute a family of ß-galactoside binding proteins some of them known to be involved in colorectal tumorigenesis. Our previous work suggests that sodium butyrate can induce galectin-12 expression in CRC cell lines. How this might be exerted is largely unexplored. In the present study we examined the potential epigenetic regulation of galectin-12 expression in colorectal cancer cell lines and primary tumors.

Material and Methods: For DNA-methylation analysis 4 microsatellite unstable (MSI) and 5 microsatellite stable (MSS) human CRC cell lines were treated with 5-Aza-2`deoxycytidine (5-Aza-dC) for 72h. Changes in galectin-12 expression were evaluated by RT-PCR analysis. Galectin-12 promoter methylation was determined by bisulfite treatment and genomic DNA sequencing of single clones in the CRC cell line SW707 as well as in FFPE specimens from 10 primary colorectal tumors (MSI: n = 5; MSS: n = 5).

Results: Treatment with 5-Aza-dC led to de novo expression of galectin-12 transcript in 4/9 (44%) of colorectal cancer cell lines. We identified a CpG island in the galectin-12 promoter region that was found to be hypomethylated upon 5-Aza-dC treatment in SW707 cells and to be associated with concomitant de novo galectin-12 mRNA expression. When primary tissues were examined we observed galectin-12 promoter hypomethylation in healthy colon mucosa but significant hypermethylation of this promoter region in corresponding tumor tissue.

Conclusions: This study for the first time shows that galectin-12 promoter methylation represses galectin-12 expression in colorectal tumor cells independent of their MSI status. In CRC cell lines this effect can be reversed by butyrate but in human colon primary tumors might be refractory to these effects of butyrate. Re-expression of galectin-12 may be a potential therapeutic strategy to constrain colorectal tumor growth.

Citation Format: Eva-Maria Katzenmaier, Johannes Gebert, Matthias Kloor, Jürgen Kopitz. Epigenetic silencing of galectin-12 in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1070. doi:10.1158/1538-7445.AM2015-1070
Abstract 108: Inhibition of demethylase, JMJD3 sensitizes diffuse large B-cell lymphoma (DLBCL) to chemotherapy
Authors: Mathur Rohit , Sehgal Lalit , Berkova Zuzana , Neelapu Sattva S. , Samaniego Felipe
Published: August 2015

Introduction

Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma (NHL) representing more than 30% of new cases diagnosed in the United States. DLBCL comprises multiple biologically and clinically distinct subtypes including germinal center B-cell-like (GCB) and activated B-cell like (ABC) DLBCL. Despite improvement in current therapies, nearly 40% of patients die from the disease. Recent studies of the epigenome and the methylome in particular, have revealed widespread epigenetic changes in DLBCL. Aberrant methylation of core histone tails occur with deregulation of methylase enzymes such as Ezh2 in DLBCL. In contrast, the role of JMJD3 and UTX, H3K27(me)3 demethylases in DLBCL remains unknown. In this study, we have evaluated the activity of the selective JMJD3 inhibitor GSK-J4 in DLBCL and investigated the mechanism of its action.

Methods

Panel of DLBCL cell lines: DB, KARPAS 422, Pfeiffer, Toledo, Sudhl2, Sudhl6, Sudhl8, TMD8, OCI-Ly1, OCI-Ly3, OCI-Ly7, OCI-Ly8, OCI-Ly4, OCI-Ly10, and primary DLBCL cells were analyzed for the expression of lysine demethylases (KDMs) using RT-PCR and Western blot analysis. Sub-G1 analysis was performed to analyze drug-induced apoptosis. Chemical inhibitor GSK-J4 and siRNAs were used for specific inhibition of the JMJD3.

Results

JMJD3 inhibitor, GSK-J4 induced the killing of DLBCL cell lines of both GC and ABC subtypes. Cell sensitivity to GSK-J4 could not be predicted from the expression of prognostic markers in DLBCL such as Bcl6, IRF4 and Ezh2. For further studies, we selected a cell line Sudhl6 (Bcl6 dependent), sensitive to the killing effect of GSK-J4. Treatment with GSK-J4 reduced the Bcl6 levels along with concomitant increase in IRF4 and FBXO11, negative regulators of Bcl6. Knockdown of JMJD3 by siRNAs also reduced the levels of Bcl6, implicating Bcl6 in the mechanism of action of GSK-J4. Additionally, GSK-J4 synergized in killing with vincristine, doxorubicin as well as proteasome inhibitors bortezomib and carfilzomib.

Conclusion

We demonstrate the potent effects of inhibition of H3K27(me)3 demethylase JMJD3 by GSK-J4 on proliferation and apoptosis of DLBCL cells. Our results clearly show synergistic effects of inhibition of demethylase with standard drugs in elimination of DLBCL cells.

Note: This abstract was not presented at the meeting.

Citation Format: Rohit Mathur, Lalit Sehgal, Zuzana Berkova, Sattva S. Neelapu, Felipe Samaniego. Inhibition of demethylase, JMJD3 sensitizes diffuse large B-cell lymphoma (DLBCL) to chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 108. doi:10.1158/1538-7445.AM2015-108
Abstract 1092: Interpreting and navigating the TCGA in the context of the RAS pathway
Authors: Yi Ming , Kessing Bailey , Stephens Robert
Published: August 2015

Analysis of large cancer data sets such as “The Cancer Genome Atlas (TCGA)” and the “International Cancer Genome Consortium (ICGC) “ has demonstrated the involvement of members of the RAS signaling pathway across many different tumor types. In particular, KRAS mutations occur at high frequency in lung, colorectal and particularly pancreatic tumors. Here, we report an analysis of TCGA data examining several aspects of KRAS and RAS pathway biology in the context several of the different data types collected through TCGA. The findings include:

• A survey of the expression of the KRAS alternatively spliced isoforms across all of the available tumor types

• Enrichment analysis from differentially expressed genes from high and low expressing samples for the 4A isoform (or the 4A/4B ratio)

• Survival analysis for RAS pathway genes in the context of mutational and expression level status

• Analysis of mutations in genes from the RAS pathway across samples and tumor types Together, these findings demonstrate that large data sets like TCGA can be used to reveal novel insights into the function of key genes that play a role in the development, maintenance and progression of cancer.

Citation Format: Ming Yi, Bailey Kessing, Robert Stephens. Interpreting and navigating the TCGA in the context of the RAS pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1092. doi:10.1158/1538-7445.AM2015-1092
Abstract 1098: Epistasis analysis of the PTHLH region in European subjects of the iCOGS breast cancer GWAS suggest multiple genes may be implicated in its role in breast cancer
Authors: Freeman Adam N. , Henderson Michael A. , Martin T John , Makalic Enes , Kapuscinski Miroslaw K. , Schmidt Daniel , Hopper John
Published: August 2015

Parathyroid hormone-related protein (PTHrP), the product of the PTHLH gene, has long been implicated in breast cancer. Its expression is thought to favour and, potentially, facilitate metastasis to bone. Paradoxically, a prospective clinical study clinical suggests that its production in primary breast cancers is actually protective, affording a better prognosis than its absence. Multiple recent Genome-Wide Association Studies (GWAS) have confirmed a single susceptibility locus immediately upstream of the PTHLH gene to be associated with breast cancer. This was again reproduced in the recent iCOGS GWAS, which involved over 90,000 European subjects. In spite of a single SNP, rs10771399, being uniquely reported and reproduced by prior studies, comprehensive analysis of the region reveals multiple significant SNPs stretching for hundreds of kbp across the locus. Epistasis analysis of this region versus the entire iCOGS array was performed using Plink!. These results suggest multiple genes may be implicated in the causation of this signal. Many known functional partners of PTHLH were identified, including IPO8, RUNX1, RUNX3, S100P, SOX9, SOX11, and SOX14. Multiple putative functional partners of PTHLH were likewise identified, including PBXIP1, PMVK, KCNN3, STX12, PPP1R8, CAMTA1, PRKCZ, TGIF1, SPHKAP, MYC, and STAT3. When taken together, these results offer novel potential insights into the function of the locus at PTHLH in breast cancer that may be further investigated.

Citation Format: Adam N. Freeman, Michael A. Henderson, T John Martin, Enes Makalic, Miroslaw K. Kapuscinski, Daniel Schmidt, John Hopper. Epistasis analysis of the PTHLH region in European subjects of the iCOGS breast cancer GWAS suggest multiple genes may be implicated in its role in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1098. doi:10.1158/1538-7445.AM2015-1098
Abstract 1100: Functional genomics to investigate the genetic determinants of cell death induced by oxidative stresses
Authors: Ding Chien-Kuang C. , Tang Xiaohu , Kim So Young , Chi Jen-Tsan A.
Published: August 2015

Increased oxidative stress in the tumor microenvironments is a prominent stress that many tumor cells need to cope with during oncogenesis. The stress depletes intracellular glutathione, increases reactive oxygen species, and finally triggers cell death in tumors. Despite its importance during oncogenesis, the genetic determinants and the cell death mechanisms under oxidative stress remain poorly defined. Here, we applied a genome-wide siRNA screen to oxidative-stress-induced cell death. Candidate genes were identified if the silencing could rescue cell death under oxidative stresses. The identified candidate genes are prioritized by the magnitude of rescue and the consistency among different siRNAs that target the same gene. Selected top hits were further functionally validated using independent siRNAs and chemicals in multiple cancer cell types. To further understand the functional aspects of these genes, we also analyzed the transcriptional and metabolomic response to oxidative stresses. By these functional genomics approach, we found that: 1) The Integrated Stress Response (ISR) is provoked by oxidative stresses to mediate cell death. Therefore, inhibition of IRE1-splicing activity, which is one of the major branches of ISR pathway, could abolish oxidative-stress-induced cell death. 2) Surprisingly, inhibition of multiple component of mTORC1 signaling, including mTOR itself, could rescue oxidative-stress-induced cell death. While further investigation is still in progress, these preliminary findings unravel a unique molecular process in which oxidative stress mediate cell death and the adaptive mechanism by which tumor cells survive these stresses.

Citation Format: Chien-Kuang C. Ding, Xiaohu Tang, So Young Kim, Jen-Tsan A. Chi. Functional genomics to investigate the genetic determinants of cell death induced by oxidative stresses. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1100. doi:10.1158/1538-7445.AM2015-1100
Abstract 1107: In whole-exome sequencing analysis, alternations of P-selectin glycoprotein ligand 1 (SELPLG) significantly associates with inflammation and survival outcomes
Authors: Cho Sunghoon , Lee Chansu , Choi Hyun , Ahn Kwang-Sung , Koh Yongil , Yoon Sung-Soo
Published: August 2015

SELPLG is type I transmembrane receptor found on the surface of neutrophils, monocytes, and most lymphocytes. SELPLG interacts with P -selectin mediates the tethering and rolling of neutrophils, monocytes, and/or lymphocytes on endothelial cells. In whole exome sequencing analysis, we found that somatic mutation of SELPLG (2%) was found. Also its alternation (37%) was found in germline control group. rs201851784 of SELPLG polymorphism (43%) was determined in 118 adult AML patients and its polymorphism (12%) was determined in 1000 genome. The prevalence of the rs201851784 polymorphism was significantly different between AML patients and normal control (AA genotype vs. GG + AG genotype). Comparison analysis of SELPLG showed X-squared = 208.4140, df = 1 and p < 2.2e-16. After the IL-6 expression levels among AML patients were detected by ELISA, the allelic status and IL-6 expression on bone marrow serum of AML patients were analyzed. AML patient having A/G alternation was found to be associated with lower levels of IL-6 compared with AML patients having A/A. Those with persistence had higher IL-6 expression levels compared with those with CR in 1st induction (P < 0.05). In addition, there is a significant correlation between levels and SELPLG rs201851784, as analyzed by linear correlation assay. The AA genotype correlated with higher IL-6 levels (P < 0.05). Reverse correlation between SELPLG rs201851784 and IL-6 levels was found. SELPLG rs201851784 and IL-6 levels were also significantly correlated with OS. In conclusion, a polymorphism in the SELPLG gene related to cell dissemination and tissue infiltration is shown for the first time to be associated with susceptibility of AML and the overall survival.

Citation Format: Sunghoon Cho, Chansu Lee, Hyun Choi, Kwang-Sung Ahn, Yongil Koh, Sung-Soo Yoon. In whole-exome sequencing analysis, alternations of P-selectin glycoprotein ligand 1 (SELPLG) significantly associates with inflammation and survival outcomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1107. doi:10.1158/1538-7445.AM2015-1107
Abstract 113: Defining the metabolic regulation of epigenetics using chemical proteomics
Authors: Meier Jordan L. , Montgomery David C. , Sorum Alexander W. , Kulkarni Rhushikesh A.
Published: August 2015

Substantial evidence indicates that chromatin modifiers are sensitive to changes in the cellular concentration of metabolites and cofactors. These findings potentially connect metabolism and gene expression, and have major implications for the regulation of cell signaling in cancer. However, missing from many of these studies has been the direct identification of the specific chromatin modifiers that sense change in metabolic state. Their identification is critical to our basic understanding of how metabolism regulates epigenetically-driven phenotypes, and may inform novel strategies to restore balance in metabolically-perturbed cancers. Towards this goal, here we report a strategy to directly define the metabolic regulation of epigenetic signaling using chemical proteomics. Our initial efforts have focused on understanding the metabolic regulation of lysine acetyltransferases (KATs), a major class of chromatin modifiers responsible for protein acetylation. We demonstrate that chemoproteomic probes can report on the interaction of acyl-CoA metabolites with KAT active sites, and when combined with affinity purification and LC-MS/MS analysis, highlight an expanded landscape of acetyltransferases present in the human genome. Next-generation chemoproteomic methods highlight the interactions of metabolic acyl-CoAs with megadalton lysine acetyltransferase protein complexes, which have implications for the regulation of KAT activity by cellular acyl-CoA pools. By defining the molecular links connecting metabolism and gene expression,these approaches will illuminate our understanding of cancer biology and inspire new therapeutic strategies to combat cancer.

Citation Format: Jordan L. Meier, David C. Montgomery, Alexander W. Sorum, Rhushikesh A. Kulkarni. Defining the metabolic regulation of epigenetics using chemical proteomics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 113. doi:10.1158/1538-7445.AM2015-113
Abstract 1142: COP1 functions as a sensor of cellular lipids and regulator of lipogenesis in human cancer cells
Authors: Huang Chenfei , Bu Yiwen , Cao Deliang
Published: August 2015

Background

Constitutive photomorphogenesis protein 1 (COP1; also known as Rfwd2), an evolutionally conserved E3 ubiquitin ligase, has been defined to mediate the degradation of several important proteins, such as p53, c-Jun, ETS, FOXO1 and ACC1, being involved in tumorigenesis. In response to DNA damage, COP1 is autodegraded in the context of ATM, which is triggered by 14-3-3σ. However, the biological function and expression regulation of COP1 remain to be fully understood. This study reports a novel function of COP1 in lipid metabolism.

Methods

We targeted COP1 expression in cancer cells MCF-7 and HepG2 with COP1 shRNA lentivirus and COP1 overexpression lentivirus, respectively. We investigated the lipid synthesis, glucose oxidation and conversion into lipids in these cells using radiolabeled acetate and glucose and lipid complex analysis. Quantitative real-time PCR and Western blot were used to examine gene expression. Acetyl CoA carboxylase-1 (ACC1) siRNA and TOFA, an inhibitor of ACC1, were used to modulate the intracellular lipid synthesis.

Results

Lipid depletion induced by acetyl CoA carboxylase-1 (ACC1) knockdown or the inhibition of ACC1 by TOFA induced COP1 expression, but palmatic acid, the end-product of de novo fatty acid synthesis pathway, reversed TOFA-induced COP1 expression. In addition, U73122, an inhibitor of phospholipase C that cleaves PIP2 into DAG and IP3, triggered COP1 expression. These data suggest that COP1 expression is mediated by cellular lipid state, and the lipid second messengers (PIP2 and DAG) are involved in the expression regulation of COP1. In contrast, the ectopic expression of COP1 greatly reduced pAMPK and pACC1 levels, but elevated fatty acid synthase (FASN) expression, leading to the increase of fatty acid (FA)/lipid synthesis. In sharp contrast, siRNA-mediated COP1 knockdown in cancer cells significantly increased pACC1 and pAMPK levels and reduced FASN expression, leading to decrease of lipid synthesis. The cell growth and proliferation was accordingly changed in response to the alterations of lipid synthesis. Altogether our data suggest that COP1 expression is regulated through the lipid second messenger mediated signaling, and the upregulated COP1 in turn enhances lipid synthesis, functioning as a positive feedback.

Conclusion

COP1 functions as a sensor of cellular lipid state and promoter of lipogenesis, facilitating tumorigenesis.

Note: This abstract was not presented at the meeting.

Citation Format: Chenfei Huang, Yiwen Bu, Deliang Cao. COP1 functions as a sensor of cellular lipids and regulator of lipogenesis in human cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1142. doi:10.1158/1538-7445.AM2015-1142
Abstract 1145: The TDP-43 confers resistance to metabolic stress condition via activating autophagy in GBM cells
Authors: Lin Tzu-Wei , Wu Chen-Wen
Published: August 2015

Glioblastoma (GBM) is the most aggressive malignant brain tumor in adults. TDP-43 (TARDBP), a DNA and RNA binding protein, is well known to play a key role in RNA processing, including involving in the pathogenesis of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Recent reports suggested that TDP-43 participate to the regulation of metabolic glycolysis in cancer. Overcoming metabolic stress is critical for cancer cell survival and growth in nutrient deprivation condition. Herein we found protein level of TDP-43 was increased in nutrient deprivation. Overexpression TDP-43 promotes glioblastoma cell survival and suppressed the activated-form levels of caspase 3 protein under nutrient deprivation. Overexpression of TDP-43 increased the tumorigenicity as determined by anchorage-independent growth assay and xenografts in immunocompromised mice model. Under nutrient deprivation, we also found that TDP-43-overexpression induced autophagy. Recent evidence suggests that autophagy is a cell survival mechanism in cancer cells. Finally, treatment of autophagy inhibitor blocks the survival of TDP-43 overexpressing GBM cells in nutrient deprivation condition. By this study, we presume TDP-43 enhanced GBM cells tumorigincity, activated autophagy and inhibited cell death under nutrient deprivation condition. TDP-43 is potentially the new aspects for understanding the tumorigenicity and metablolic regulation for malignant brain tumors and GBM.

Citation Format: Tzu-Wei Lin, Chen-Wen Wu. The TDP-43 confers resistance to metabolic stress condition via activating autophagy in GBM cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1145. doi:10.1158/1538-7445.AM2015-1145
Abstract 1187: Functional genomic screening reveals asparagine dependence as a metabolic vulnerability in sarcoma
Authors: Hettmer Simone , Schinzel Anna C. , Tchessalova Daria , Richards Nigel GJ , Hahn William C. , Wagers Amy J.
Published: August 2015

Background. Currently available therapies for sarcomas are often inadequate. This study sought to identify actionable gene targets in sarcomas by selective targeting of the molecular networks that support sarcoma cell proliferation. Experimental approach. Mouse sarcomas driven by expression of oncogenic Kras(G12v) and disruption of CDKN2A (p16p19) were induced by ex-vivo transduction and intramuscular injection of mouse satellite cells. Kras; p16p19null sarcomas identified a cluster of genes upregulated in mouse sarcomas and human rhabdomyosarcoma compared to normal skeletal muscle. A customized shRNA proliferation screen was used to identify transcripts within this cluster that reduced sarcoma cell proliferation. Target gene effects on sarcoma growth were evaluated in mouse and human sarcoma cell lines and xenografts.

Results. Silencing of asparagine synthetase (ASNS), an amidotransferase that converts aspartate into asparagine, produced the strongest inhibitory effect on sarcoma growth in a functional genomic screen of mouse sarcomas generated by oncogenic Kras and Cdkn2a. ASNS silencing in mouse and human sarcoma cell lines reduced the percentage of S phase cells and impeded new polypeptide synthesis. These effects of ASNS silencing were reversed by exogenous supplementation with asparagine. Also, asparagine depletion via the ASNS inhibitor amino sulfoximine 5 (AS5) or asparaginase inhibited mouse and human sarcoma growth in vitro, and genetic silencing of ASNS in mouse sarcoma cells combined with depletion of plasma asparagine inhibited tumor growth in vivo. Conclusions. The generation of new protein mass by rapidly proliferating sarcoma cells requires adequate Asparagine availability. Asparagine reliance of sarcoma cells may represent an actionable, metabolic vulnerability with potential anti-rhabdomyosarcoma therapeutic value.

Citation Format: Simone Hettmer, Anna C. Schinzel, Daria Tchessalova, Nigel GJ Richards, William C. Hahn, Amy J. Wagers. Functional genomic screening reveals asparagine dependence as a metabolic vulnerability in sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1187. doi:10.1158/1538-7445.AM2015-1187
Abstract 1190: Prognostic significance of serine synthesis pathway-related protein expression in patients with resected colon cancer
Authors: Kang Byung Woog , Kim Jong Gwang , Chae Yee Soo , Lee Soo Jung , Yoon Shinkyo , Yoon Ghil Suk
Published: August 2015

Background

We analyzed the prognostic impact of serine synthesis pathway-related protein expression in a large number of patients with colon cancer.

Methods

PDK1, PDK2, PKM2, PHGDH, PSAT, and ER betta expression were investigated on the basis of the immunohistochemistry of tissue microarray specimens from 486 colon cancer patients who underwent curative surgery between 2006 and 2010. We scored the staining intensity (0 through 3) and percentage of positive cells (0 through 4), and the staining score was defined as sum of these scores used to categorize the each expression as low (0 through 3) or high (4 or more).

Results

For the 486 patients, the median age was 66 (30-90) years at the time of surgery. The pathologic stages after the surgical resection were as follows: stage I (n = 98), stage II (n = 213), and stage III (n = 175). A total of 201 (41.4%) was identified as high PDK2 (staining score ≥4). The survival analysis showed that the expression of serine synthesis pathway-related protein had no effect on the overall survival and disease-free survival (DFS) except that the grade of PDK2 expression was significantly associated with the DFS (p = 0.028). On the multivariate analysis that included age, histology, TNM stage, and CEA level, no significant association between the grade of the PDK2 expression and DFS was observed.

Conclusion

The current study suggests that the tissue expression of serine synthesis pathway-related protein is not an independent prognostic marker for patients with surgically resected colon cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Byung Woog Kang, Jong Gwang Kim, Yee Soo Chae, Soo Jung Lee, Shinkyo Yoon, Ghil Suk Yoon. Prognostic significance of serine synthesis pathway-related protein expression in patients with resected colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1190. doi:10.1158/1538-7445.AM2015-1190
Abstract 1203: Metastasis-associated oncogene RhoC as a regulator of glutamine metabolism in the inflammatory breast cancer cell line SUM149
Authors: Yates Joel A. , Wynn Michelle L. , Wu ZhiFen , Evans Charles R. , Burant Charles , Schnell Santiago D. , Merajver Sofia D.
Published: August 2015

Metabolic reprogramming is increasingly recognized as a fundamental hallmark of cancer. While the Warburg effect and normal proliferative metabolism are similar, they are not equivalent. We hypothesize that there are key drivers of malignant metabolism that can be modulated to impede cancer proliferation without substantial effects on normal tissue growth. Using 13C-labeled glucose and glutamine tracers in combination with mass spectrometry and measurements of extracellular glucose, lactate, and glutamine flux, we have characterized system level differences in a series of breast cancer cell lines as well as normal-like breast epithelial cells. We observed an increase in the reductive carboxylation of glutamine-derived citrate and alpha-ketoglutarate in the triple-negative inflammatory breast cancer cell line SUM149. We also observed that the SUM149 exhibit high levels of HIF-1α and low levels of oxygen consumption under normoxia, suggesting that the cell line is highly adapted to hypoxia. Surprisingly, the stable depletion of HIF-1α via shRNA had no significant effect on the metabolic profile of these cells. Previous work by our lab and others has demonstrated that the GTPase RhoC is a driver of the metastatic phenotype exhibited by inflammatory breast cancer. Activation of RhoC is known to induce cytoskeletal rearrangements and increase invasive potential. The Rho GTPase family of proteins has also recently been linked to metabolism, specifically regulation of glutaminase activity. Here we show that stable knockdown of RhoC in SUM149 cells results in a marked decrease in the rate of both glutamine uptake and intracellular reductive carboxylation. This work reinforces the role of RhoC as an important driver of inflammatory breast cancer metastatic potential. We conclude that RhoC remains an important clinical target with the potential to alter patient outcomes.

Citation Format: Joel A. Yates, Michelle L. Wynn, ZhiFen Wu, Charles R. Evans, Charles Burant, Santiago D. Schnell, Sofia D. Merajver. Metastasis-associated oncogene RhoC as a regulator of glutamine metabolism in the inflammatory breast cancer cell line SUM149. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1203. doi:10.1158/1538-7445.AM2015-1203
Abstract 1209: Dissecting by 13C tracers the utilization of glucose and glutamine for the synthesis of fatty acids in triple negative breast cancer cells
Authors: Strigun Alexander , Reszka Regina , Schiewe Hans-Joerg , Laine Jean-Philippe , Rennefahrt Ulrike
Published: August 2015

Triple negative breast cancer (TNBC) is an aggressive cancer, which is resistant to the majority of available chemotherapies. We have recently identified significant differences in the levels of membrane lipids and fatty acids between two distinct TNCB metabolic subtypes (MST2 and MST3) (Beatty et al., 2014, manuscript submitted). Fatty acid synthesis has recently been discussed as potential pharmaceutical target.

In order to evaluate whether glucose or glutamine is the preferred carbon source for palmitic acid synthesis, TNBC cell lines (BT549 and HCC1806) were fed with fully labelled glucose and fully labelled glutamine ([U-13C6]glucose and [U-13C5]glutamine) in independent experiments. Fully labelled glutamine allows conclusions on total usage of glutamine via the reductive and the oxidative part of the TCA-cycle. In order to assess the contribution of glutamine to palmitic acid synthesis via the reductive pathway we fed the subtypes with single labelled glutamine [5-13C1]glutamine. Enrichment of 13C in palmitic acid from all labelling experiments was measured via GC-MS (gas chromatography coupled mass spectrometry).

Feeding the TNBC subtypes with [U-13C6]glucose and [U-13C5]glutamine resulted in a higher enrichment of 13C in palmitic acid derived from glucose as compared to glutamine in both subtypes, indicating that glucose is the main carbon source at the tested conditions. Feeding the cells with [5-13C1]glutamine also resulted in an enrichment of 13C in palmitic acid in both subtypes, which indicates that reductive glutamine metabolism contributes to palmitic acid synthesis in both subtypes. Based on the 13C-palmitic acid data from both glutamine isotopes we found that palmitic acid synthesis preferably occurs via the reductive path in MST3 (61% of total glutamine use) while the oxidative path seems the preferred route (34% of total glutamine use) in MST2.

The presented data indicate that glucose is the main carbon source for fatty acid synthesis in both MSTs, suggesting a higher sensitivity towards pharmacological targeting of glucose metabolism. Despite of the lower dependency on glutamine, TNCBs of the MST3 subtype seem more sensitive towards targeting the reductive glutamine metabolism.

Citation Format: Alexander Strigun, Regina Reszka, Hans-Joerg Schiewe, Jean-Philippe Laine, Ulrike Rennefahrt. Dissecting by 13C tracers the utilization of glucose and glutamine for the synthesis of fatty acids in triple negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1209. doi:10.1158/1538-7445.AM2015-1209