bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2148: Ras-mediated evasion of detachment-induced cell death involves differential signaling pathways for metabolism and anoikis
Authors: Mason Joshua A. , Versagli Calli , Leliaert Amy , Durbin Sienna , Buchheit Cassandra , Schafer Zachary
Published: August 2015

In order for successful metastasis to occur, cells must overcome anoikis, a caspase-dependent cell death process triggered by detachment from the extracellular matrix (ECM). In addition, recent studies have revealed that ECM-detached cells must also rectify detachment-induced metabolic defects that compromise cell survival. However, the precise signals involved in the inhibition of anoikis and the restoration of proper cell metabolism during ECM-detachment are poorly understood. Of particular interest to our studies is the oncogene Ras, which is constitutively active in approximately 30% of all cancers and is well known to be regulate cell death pathways and metabolism. We have discovered that Ras facilitates the survival of ECM-detached cancer cells by utilizing distinct signaling pathways to block anoikis and regulate metabolism.

Using MCF-10A cells engineered to overexpress oncogenic Ras and HCT116 cells (which contain an activating Ras mutation), we investigated the signaling pathways downstream of Ras that facilitate the survival of ECM-detached cells. Interestingly, we discovered that while Ras-mediated PI(3)K signaling is critical for rectifying metabolic defects during ECM-detachment, the downstream effector is not Akt, but rather SGK-1. SGK-1 stimulates glucose uptake, enhances ATP generation, promotes luminal filling in 3-dimensional cell culture, and drives anchorage-independent growth in soft agar. Interestingly, our data also indicate that oncogenic Ras utilizes an entirely distinct signaling pathway to block anoikis. We discovered that Ras diminishes the expression of the phosphatase PHLPP1. This inhibits the dephosphorylation-induced activation of a signaling cascade that culminates in the activation of pro-apoptotic p38 MAPK. In aggregate, these data unveil a novel survival strategy utilized by ECM-detached cancer cells and implicate both SGK-1 and PHLPP1 function downstream of Ras during ECM-detachment. The molecular mechanisms unveiled here could be utilized for the design of novel therapies that eliminate ECM-detached, metastatic cancer cells with Ras mutations through simultaneous modulation of SGK-1 and PHLPP1.

Citation Format: Joshua A. Mason, Calli Versagli, Amy Leliaert, Sienna Durbin, Cassandra Buchheit, Zachary Schafer. Ras-mediated evasion of detachment-induced cell death involves differential signaling pathways for metabolism and anoikis. [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 2148. doi:10.1158/1538-7445.AM2015-2148
Abstract 2151: Development of reference reagents to accelerate research on the RAS pathway
Authors: Esposito Dominic
Published: August 2015

The Ras Reference Reagents program within the NCI national Ras mission is designed to produce and distribute qualified DNA vectors, cell lines, and viruses to supply to the external community to facilitate research into Ras biology and development of Ras cancer therapeutics. Initial sets of reagents include sequence-verified libraries of Ras mutants and Ras pathway genes in Gateway combinatorial cloning vectors in multiple formats. An associated library of combinatorial components is also available to permit simple generation of expression vectors with various promoters, fusion tags, and plasmid backbones. Additional reagents under construction validated plasmid and lentiviral reagents for shRNA and CRISPR delivery, qualified cell lines, and systems for the generation of highly purified and characterized Ras and Ras-related proteins. All of these resources are readily available to the academic community through simple mechanisms, and can be licensed to industry as well. The Ras Reference Reagents program is also interested in identifying the needs of the community for additional Ras-related resources, and is developing methods for vetting requests from the outside to provide additional reagents.

Citation Format: Dominic Esposito. Development of reference reagents to accelerate research on 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 2151. doi:10.1158/1538-7445.AM2015-2151
Abstract 2156: FGFR2 regulates Mre11 expression and double-strand break repair via the MEK-ERK-POU1F1 pathway in breast tumorigenesis
Authors: Huang Yuan-Ling , Chou Wen-Cheng , Hsiung Chia-Ni , Hu Ling-Yueh , Chu Hou-Wei , Shen Chen-Yang
Published: August 2015

Purpose: The association between breast cancer risk and genetic variants of fibroblast growth factor receptor 2 (FGFR2) has been identified and repeatedly confirmed; however, the mechanism underlying FGFR2 in breast tumorigenesis remains obscure. Given that breast tumorigenesis is particularly related to DNA double-strand-break-repair (DSBR), we examined the hypothesis that FGFR2 is involved in DSBR.

Experimental design: This study started with the demonstration that FGFR2 impaired DSBR. Subsequent screening of the expression of genes functionally related to DSBR was performed and possible signaling pathways downstream of FGFR2 were examined. Data from independent breast cancer patient cohorts was used to confirm that difference in progression of patients can be explained by the findings identified in cell model.

Results: Expression of Mre11, a vital exonuclease in DSBR, is downregulated by FGFR2, which is further linked to decreased DSBR. Analysis of the Mre11 promoter revealed that POU1F1 mediates FGFR2-induced Mre11 downregulation. Furthermore, ERK, downstream of FGFR2, directly interacts with and phosphorylates POU1F1, increasing POU1F1 binding capacity to the Mre11 promoter and repressing Mre11 expression, which consequently affects DSBR and sensitizes breast cancer cells to chemotherapeutic treatments. The importance of the FGFR2-Mre11-DSBR link in cancer progression is suggested by the finding that genotypes of FGFR2 and Mre11 are associated with survival of breast cancer patients and that FGFR2 expression correlates with cancer prognosis specifically in patients receiving chemotherapy.

Conclusion: This study yields important insight into the role of FGFR2 in breast tumorigenesis and may facilitate development of a useful therapeutic approach for breast cancer. With the move toward personalized therapy, which has gradually become a clinical reality, the findings of the present study suggest that FGFR2 expression may be a potential biomarker for selecting a suitable subgroup of breast cancer patients to receive chemotherapy or to determine the optimal chemotherapeutic dose.

Citation Format: Yuan-Ling Huang, Wen-Cheng Chou, Chia-Ni Hsiung, Ling-Yueh Hu, Hou-Wei Chu, Chen-Yang Shen. FGFR2 regulates Mre11 expression and double-strand break repair via the MEK-ERK-POU1F1 pathway in breast tumorigenesis. [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 2156. doi:10.1158/1538-7445.AM2015-2156
Abstract 2157: PI3K/AKT-induced stabilization of FUSE binding proteins (FBPs) in liver cancer cells
Authors: Samarin Jana , Stein Ilan , Horwitz Elad , Chen Xin , Malz Mona , Pikarsky Eli , Calvisi Diego , Schirmacher Peter , Breuhahn Kai
Published: August 2015

Far upstream element-binding proteins (FBPs) represent a family of transcription factors, which are highly overexpressed in the majority human liver cancer (hepatocellular carcinoma; HCC). Nuclear accumulation of FBP-1, -2, and -3 in HCC cells support cell proliferation and migration. However, the mode of FBP dysregulation in HCC cells has not been defined so far.

Genomic alterations of the fubp (FBP-1; chr. 1p31.1), khsrp (FBP-2; chr. 19.p13.3), and fubp3 (FBP-3; 9p34.11) gene loci in primary human HCCs were determined by matrix-comparative genomic hybridization (CGH). In order to identify specific stimuli of FBP enrichment, different human HCC cell lines were cultured under different cell density and hypoxic conditions or treated with growth factors (e.g., TGFß, IGF-II), chemical inhibitors (e.g., AG1478, SB203580, MEK1/2, sorafenib, and PI3K inhibitors), and gene-specific siRNAs (e.g., Akt1-3, rictor, raptor). The in vivo relevance of the findings was confirmed using HCC mouse models (e.g., Mdr2-/- animals) and human HCC tissue specimens (immunohistochemistry and western blotting).

Only few genomic gains were found for all FBP family members in human HCC samples (e.g., 6% for fubp). In vitro, inhibition of EGFR (by AG1478) or administration of the multi-kinase inhibitor sorafenib reduced FBP protein levels. Furthermore, inhibition of the PI3K/Akt/mTOR pathway revealed strong effects on FBP protein half-live without significant impact on FBP transcription. Other pathways or stimuli only caused minor effects on FBP expression (e.g., p38 or Raf1 inhibition). These findings were confirmed in mouse HCC models with high-level expression of FBPs, where injection of sorafenib or rapamycin significantly diminished FBP amounts. Reduction of FBP concentrations after inhibition of the PI3K/Akt/mTOR pathway was rescued after inhibition of caspase-3/-7 activity. Lastly, a significant correlation between pAkt and nuclear FBP expression was detected in human HCC tissues.

These results demonstrate that activation of the PI3K/Akt/mTOR signalling axis is responsible for increased stabilisation of FBPs in human HCC cells. Elevated FBP half-live is at least partly mediated through crosstalk between the PI3K/Akt/mTOR pathway and caspase activity. These data are important for a deeper understanding of how druggable upstream regulators may affect FBP bioavailability in hepatocarcinogenesis.

Citation Format: Jana Samarin, Ilan Stein, Elad Horwitz, Xin Chen, Mona Malz, Eli Pikarsky, Diego Calvisi, Peter Schirmacher, Kai Breuhahn. PI3K/AKT-induced stabilization of FUSE binding proteins (FBPs) in liver 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 2157. doi:10.1158/1538-7445.AM2015-2157
Abstract 2173: Robust estimation of mutation burden
Authors: Westesson Oscar , Nielsen Rasmus , St John John , Caulin Aleah , Hahner Nicholas , Stewart Stewart , Foo Catherine , Lung Kimberly , Catalano Jeff , Lee Mandy , Giannikopoulos Petros , Polkinghorn Will , Wiessman Jonathan , Regev Aviv , Bivona Trever
Published: August 2015

Developing a more robust approach to measure mutational burden is of central importance to improving the characterization of the molecular profile of tumor and may improve our ability to predict tumor progression or response to therapy in patients. Mutational burden is typically calculated as a direct enumeration of called somatic mutations per megabase covered. However, there is a growing appreciation that tumor purity, variable sequencing coverage, and copy number alterations can substantially impact the accurate identification any specific somatic mutation. Furthermore, population genetic theory and empirical data indicate that in many cases the vast majority of somatic mutations appear in only a small subpopulation of tumor cells, a context in which there is a high likelihood that an individual subclonal mutation may not be identified by conventional analysis. This tendency to miss low frequency mutations is highly variable and dependent, in part, upon sample purity and results in a strong source of bias not addressed in existing methods to measure variant allele frequencies.

We present a novel computational method that incorporates these sources of bias in a coherent probabilistic framework that enables maximum-likelihood inference of relevant population parameters such as mutation burden. We apply our method to simulated data as well as patient tumor samples diluted with varying known proportions of normal DNA. We show that our approach allows us to generate estimates of mutation burden that are robust to the substantial variations in purity and sequencing coverage that are frequently encountered in patient tumor analysis. Hence, our novel method may improve the accurate detection and quantification of variant alleles in patient tumors to better understand their genetic landscape and guide clinical management.

Citation Format: Oscar Westesson, Rasmus Nielsen, John St John, Aleah Caulin, Nicholas Hahner, Stewart Stewart, Catherine Foo, Kimberly Lung, Jeff Catalano, Mandy Lee, Petros Giannikopoulos, Will Polkinghorn, Jonathan Wiessman, Aviv Regev, Trever Bivona. Robust estimation of mutation burden. [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 2173. doi:10.1158/1538-7445.AM2015-2173
Abstract 2177: Differential expression of stromal cell-related genes between primary nasopharyngeal carcinoma and distant metastasis
Authors: Ke Liangru , Chen Jinna , Wang Lin , Yu Yahui , Ye Yanfang , Xia Weixiong , Lv Xing , Yang Jing , Guo Xiang , Xiang Yanqun , Guan Xinyuan
Published: August 2015

Stromal cells in the tumor microenviroment play an important role in development and progression of nasopharyngeal carcinoma (NPC). Aim of this study is to identify the stromal cell related genes which may promote distant metastasis in NPC. Materials and methods: Differently expressed genes were accessed by comparing nasopharyngeal carcinoma and distant metastasis by microarray. Then stromal cells related genes were analyzed by using statistics tools. Five selected genes were validated in 112 nasopharyngeal carcinoma specimens. Results: We have identified 3360 genes up-regulated (P < .05)and 2327 genes down-regulated(P < .05) in distant metastasis versus primary nasopharyngeal carcinoma, among the differently expressed genes,111 stromal cells related genes were picked up according to previous researches for cluster analysis and pathway analysis. Upon validating 5 selected genes (BIRC3,FRAS1,NPNT,POSTN,PCSK6) in 112 additional NPC cases, the authors found that expression of POSTN was negative correlated significantly with lymph node involvement (p = 0.006)and clinical stage(p = 0.035) at the moment of diagnosis and FRAS1 was negative correlated with disease-free survival (p = 0.002) and distant metastasis survival (p = 0.02).Conclusion: Increasing knowledge of metastasis promotion of stromal cells related genes may provide feasible explanations underlying NPC progression and metastasis along with the development of biomarkers for screening high-risk populations.

Citation Format: Liangru Ke, Jinna Chen, Lin Wang, Yahui Yu, Yanfang Ye, Weixiong Xia, Xing Lv, Jing Yang, Xiang Guo, Yanqun Xiang, Xinyuan Guan. Differential expression of stromal cell-related genes between primary nasopharyngeal carcinoma and distant metastasis. [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 2177. doi:10.1158/1538-7445.AM2015-2177
Abstract 2188: Different gene expression between men and women patients with lung adenoarcinoma reveals enrichment of immune gene sets
Authors: Pinto Joseph A. , Araujo Jhajaira M. , Prado Alexandra , Flores Claudio , Cardenas Nadezhda K. , Zaharia Mayer , Sarria Gustavo , Aguilar Alfredo , Neciosup Silvia , Gomez Henry , Mas Luis
Published: August 2015

INTRODUCTION:

There is different susceptibility to DNA damage induced by tobacco smoking between women and men. In despite of the great advances in the knowledge of the genomic landscape of lung cancer, is not explored the molecular differences between man and women. Our aim was to evaluate differentially enriched gene sets between the two genders.

METHODS:

We evaluated 03 public databases containing gene expression values from lung adenocarcinoma patients: GSE50081 (HG-U133_Plus_2; n = 181 samples), GSE47115 (Illumina HumanHT-12 WG-DASL V4.0 R2; n = 46 samples), GSE10072 (HG-U133A; n = 107 samples). We perform the Gene Set Enrichment Analysis (GSEA) to find differences between the genders. Each dataset was analyzed individually and was divided in cohorts of smokers and non-smokers (and healthy tissues by smoking status when it was included in the dataset). Cases with unknown smoking status and former smokers were excluded from the analysis. We use the Gene Ontology biological process terms to find similar enriched pathways between cohorts. We consider enriched process with statistical differences (p<0.05) or trends (P<0.08) in at least a cohort.

RESULTS:

The analysis did show immune genes enrichment in women such as cytokine production, defense response to bacteria and response to other organisms. In another hand, regulation of JNK and MAPKKK cascades was enriched in tumors and healthy tissues from non-smokers women. In men, the oxidoreductase activity acting on sulfur group of donors was enriched in smoking men.

CONCLUSION:

There are differences between tumors from women and men that should be deeply explored.

Citation Format: Joseph A. Pinto, Jhajaira M. Araujo, Alexandra Prado, Claudio Flores, Nadezhda K. Cardenas, Mayer Zaharia, Gustavo Sarria, Alfredo Aguilar, Silvia Neciosup, Henry Gomez, Luis Mas. Different gene expression between men and women patients with lung adenoarcinoma reveals enrichment of immune gene sets. [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 2188. doi:10.1158/1538-7445.AM2015-2188
Abstract 2190: Oncostatin M reduces SLUG expression in lung adenocarcinoma cells through STAT1-PIAS4-dependent histone deacetylation
Authors: Pan Chih-Ming , Wang Mong-Lien , Wu Cheng-Wen
Published: August 2015

We previously described that mesenchymal stem cells (MSCs) secrete a multifunctional cytokine - Oncostatin M (OSM) which effectively inhibited the metastasis and growth of LAC cells, and reduce the SLUG expression, leading to mesenchymal-epithelial transition (MET). Our data showed that the OSM-dependent inhibition of SLUG is mediated by a STAT1-dependent pathway through a transcriptional control. Previous studies have shown both positive and negative role of STAT1 in transcriptional regulation of its downstream target genes. However, the underlying mechanism of how STAT1 mediates the suppression of SLUG transcription remains elusive.

To investigate the associated effector or mediator in OSM-induced STAT1-dependent SLUG suppression, we knocked down PIAS4 (PIAS4-KD), a co-repressor of STAT1, in A549 cells using lentiviral expressed short hairpin RNAs (shRNA). We demonstrated that knockdown of PIAS4 reversed the OSM-suppressed SLUG level and resulted in a higher SLUG mRNA in comparison to scrambled shRNA control. Moreover, we constructed SLUG promoter-driven luciferase expression reporters and test the effect of PIAS4 on SLUG promoter activity. We observed that PIAS4-KD cells expressed higher SLUG promoter activity than control cells, and OSM treatment further augmented this difference. In contrast, reconstitution of PIAS4 expression in PIAS4-KD cells decreased the SLUG promoter activity. Chromatin immuneprecipitation assay (ChIP) revealed the binding of PIAS4 to SLUG promoter region one hour after OSM administration. We also showed OSM-induced tyrosine-phosphorylated STAT1 interacts with PIAS4 and HDAC1 in a co-immunoprecipitation assay. Finally, the level of H3K9 acetylation in SLUG promoter region decreased significantly upon OSM treatment, and the pan HDAC inhibitor, trichostatin A (TSA) rescues OSM-induced suppression of SLUG promoter activity.

Our findings demonstrated the underlying mechanism of the tumor-suppressive effect of OSM, and revealed a STAT1-PIAS4-dependent regulation of SLUG promoter through histone deacetylation.

Citation Format: Chih-Ming Pan, Mong-Lien Wang, Cheng-Wen Wu. Oncostatin M reduces SLUG expression in lung adenocarcinoma cells through STAT1-PIAS4-dependent histone deacetylation. [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 2190. doi:10.1158/1538-7445.AM2015-2190
Abstract 2192: Stress-induced reprogramming of epithelial cancers toward a stem-like fate
Authors: Gozo Maricel C. , Cheresh David A. , Yebra Mayra , Sequin Laetitia
Published: August 2015

Cancer cell plasticity has become a focus of intense study as it impacts both therapy and drug resistance. Epigenetic changes contributing to cellular plasticity and stemness allow tumor cells to adapt to microenvironmental stress and or drug treatment. We recently reported that the heterodimer integrin receptor αvβ3 serves as a marker of stem-like breast, lung, and pancreatic carcinomas that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib.  In fact, αvβ3 was found to be both necessary and sufficient to drive stemness, drug resistance, and tumor progression. Here, we report that β3 expression is expressed in response to a range of cellular stresses, including nutrient deprivation, hypoxia, oxidative stress, and cancer therapy.  Stress-induced β3 gene expression is enhanced by epigenetic histone modifications that promote an open chromatin state enabling binding of the HNF4A transcription factor to the β3 promoter.  Once expressed, β3 induces reprogramming toward a stem-like fate by driving a subset of NFκB target genes, including FOXP1, a transcription factor that controls expression of pluripotency genes OCT4 and NANOG.  Therefore, stress-induced chromatin remodeling and the HNF4A-mediated induction of β3 expression triggers a switch from an epithelial to a cancer stem-like phenotype.  Evidence is provided that pharmacological blockade of this epigenetic change not only inhibits β3 expression but reverses tumor stem-like properties and enhances their response to therapeutic standard of care.  These findings define a stress-induced but reversible epigenetic pathway leading to β3 expression on a range of epithelial cancers that drives cancer stemness, tumor progression and drug resistance.  

Citation Format: Maricel C. Gozo, David A. Cheresh, Mayra Yebra, Laetitia Sequin. Stress-induced reprogramming of epithelial cancers toward a stem-like fate. [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 2192. doi:10.1158/1538-7445.AM2015-2192
Abstract 2193: Hypoxia-induced G9a histone methyltransferase is crucial for blunting RUNX3 tumor suppressor function
Authors: Lee You Mie
Published: August 2015

Recent our work shed light on the critical roles of RUNX3 in early tumorigenesis. Hypoxia has been reported to be an early tumorigenic insult. Previously, we showed that RUNX3 is silenced by G9a HMT under hypoxia. However, the possible direct modification of RUNX3 by G9a induced by hypoxia and its outcome are not yet investigated. Here we show that RUNX3 physically interacts with G9a. Under hypoxic conditions, G9a directly methylates lysines 129 and 171 of RUNX3. G9a-methylated RUNX3 is localized to the cytoplasm and degrade through Smurf-mediated ubiquitination pathway. Hypoxia and G9a overexpression decreased RUNX3-responsive genes by DNA microarray analysis. Consistently, tumor suppressive role of RUNX3 was decreased by G9a-mediated methylation in gastric cancer xenograft model. Taken together, our results demonstrate that methylation by G9a is a key chemical modification for the regulation of RUNX3 tumor suppressor function in hypoxic conditions and in early tumorigenic stage.

Citation Format: You Mie Lee. Hypoxia-induced G9a histone methyltransferase is crucial for blunting RUNX3 tumor suppressor function. [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 2193. doi:10.1158/1538-7445.AM2015-2193
Abstract 2201: EGFR regulates the expression and activity of ARID3B through phosphoinositide 3-kinase
Authors: Hallas William M. , Cowden Dahl Karen
Published: August 2015

ARID3B is a transcription factor that is upregulated in ovarian cancer and its expression correlates with relapse following chemotherapy. However, the mechanism that leads to ARID3B's elevated expression in ovarian cancer has not been elucidated. EGFR is commonly overexpressed in ovarian cancer, as is phosphoinositide 3-kinase (PI3K), lending us to believe that this pathway may be regulating ARID3B. We demonstrated that epidermal growth factor receptor (EGFR) signaling induces expression of ARID3B through the ETS family transcription factor PEA3. However, the exact mechanism that controls EGFR induction of ARID3B has not been elucidated, and the impact of EGFR on ARID3B activity is unknown. To find the optimal time point for EGF induction of ARID3B mRNA, ovarian cancer cells were serum-starved and then treated with EGF for different amounts of time. We then examined the expression of ARID3B mRNA induced by this treatment in each case. We found that ARID3B was most strongly induced between 2 and 4 hours after treatment with EGF. We then performed dose curve experiments for the PI3K inhibitors, wortmannin and LY294002. We performed qRT-PCR to examine ARID3B mRNA expression under the different concentrations of inhibitors. Western blots for the presence of phosphorylated Akt under each concentration demonstrated the effectiveness of the inhibitors. Next, ovarian cancer cells were serum-starved and then treated with EGF or EGF in combination with PI3K inhibitors. Both immunoprecipitation and chromatin immunoprecipitation (ChIP) were performed on these samples. Western blot for phospho-serine was conducted on immunoprecipitated samples to test for serine phosphorylation of ARID3B in response to growth factor signaling. We found that ARID3B is serine phosphorylated. Next, we performed ChIP to assess the binding of ARID3B to select target genes. We found on the genes we tested, EGF treatment decreased DNA promoter binding. Binding was greatest in the serum-starved samples and the samples treated with the PI3K inhibitors. Very little binding was seen in the EGF treated cells. These results were consistent for a number of ARID3B target genes. In future studies, we will determine which serine residues are phosphorylated by doing a mass spectrometric analysis. Then, site directed mutagenesis will be conducted to test the requirement of serine phosphorylation to target gene regulation. By understanding how ARID3B functions as a transcription factor, we will gain critical insight into ovarian cancer relapse and progression.

Citation Format: William M. Hallas, Karen Cowden Dahl. EGFR regulates the expression and activity of ARID3B through phosphoinositide 3-kinase. [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 2201. doi:10.1158/1538-7445.AM2015-2201
Abstract 2202: Novel regulation of pro-migratory genes by nuclear Src in pancreatic cancer cells
Authors: Paladino David
Published: August 2015

Pancreatic cancer is an exceptionally lethal malignancy with a poor prognosis, and there is little known about the molecular mechanisms that lead to the disease and its progression. Several reports have implicated dysregulated Src tyrosine kinase function in pancreatic tumorigenesis and progression. However, except for the classic role of Src in promoting events that contribute to invasiveness, not much is known of how the tyrosine kinase supports pancreatic cancer progression and the poor response of the disease to therapy. In previous reports, we showed that Src is present in the cell nucleus of pancreatic cancer cells in association with gene promoter sequences. Mass spectrometry analysis of Src co-immune complex from nuclear lysate preparations from pancreatic cancer cells identified p300 as a novel Src-interacting protein. Chromatin immunoprecipitation coupled to human promoter microarray (ChIP-chip) analysis showed that nuclear Src is associated with the promoters of SMYD3 and HMGA2 in pancreatic cancer cells. Inhibition of Src tyrosine kinase or p300 lysine acetyltransferase activity decreased the expression of SMYD3 and HMGA2 mRNA and protein levels in pancreatic cancer cells in a time-dependent manner and blocked pancreatic cancer cell migration. Current data altogether show that Src at the inner nuclear envelope is physically associated with and regulates the expression of SMYD3 and HMGA2, through interaction with p300. The studies identify a novel Src function through epigenetic regulation of pro-metastatic genes and provide additional potential avenues for developing new treatment strategies for metastatic pancreatic cancer.

Citation Format: David Paladino. Novel regulation of pro-migratory genes by nuclear Src in pancreatic 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 2202. doi:10.1158/1538-7445.AM2015-2202
Abstract 2224: Functional role of NRF2 signaling in sphere cultured carcinoma cells
Authors: Ryoo In-geun , Kwak Mi-Kyoung
Published: August 2015

Cancer stem cells (CSCs) are known to express high levels of antioxidant proteins and ABC transporters. In this study, we investigated the potential involvement of NF-E2-related factor 2 (NRF2) in CSC survival and stress response by using the MCF7 mammosphere culture, which is accepted as a CSCs-enriched system. The levels of NRF2 protein, reporter transcription activity, and target gene expression such as efflux transporters were higher in mammospheres than in the monolayer. We observed that proteasome activity as well as catalytic subunits expression was decreased in the mammospheres. Additionally, mammospheres showed an elevated p62 protein level and p62-knockdown suppressed NRF2 activation. The role of NRF2 in mammospheres could be demonstrated in NRF2-knockdown mammospheres. Sphere growth was retarded in knockdown group and anticancer drugs resistance was not obtained in NRF2-knockdown mammospheres in contrast to the control mammospheres. NRF2 activation could be confirmed in spheres-culture of colon cancer and ovarian cancer cells. These results show that reduced proteasome function and elevated p62 level contributed to NRF2 activation in CSCs-enriched mammospheres. The study further suggests a potential role of NRF2 in CSCs survival and stress resistance.

Citation Format: In-geun Ryoo, Mi-Kyoung Kwak. Functional role of NRF2 signaling in sphere cultured carcinoma 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 2224. doi:10.1158/1538-7445.AM2015-2224
Abstract 2226: PLSCR1- a modifier of TGFβ signaling
Authors: Klauzinska Margaret , Karasawa Hideaki , Rangel Maria Cristina , Castro Nadia , Baker Alyson , Bertolette Daniel , Krask Julie , Salomon David
Published: August 2015

The PLSCR family has been proposed to play a role in the redistribution of plasma membrane phospholipids. Recent work on the PLSCRs has revealed their additional functions such as signal transduction and lipid metabolism. The most studied member of this family, PLSCR1, has been shown to partition into lipid rafts implicating it in the regulation and endocytic trafficking of receptor-signaling complexes, e.g. EGFR. Additionally, PLSCR1 has been implicated in tumorigenesis although the exact mechanism of PLSCR1 is still controversial.

In a previous study, we identified PLSCRs, as novel Cripto-1 (CR-1) binding proteins that might modulate CR-1-dependent signaling pathways. In this study, we confirmed that PLSCR1 and PLSCR3 bind to Cripto-1 but also that PLSCR1 also inhibits TGFβ signaling via binding to the receptor complexes for the TGFβ superfamily. We also showed that PLSCR1 mediates protein degradation of type I receptors: Alk4 and TGFβRI but not ActRBII, TGFBRII or BMPRb.

Additionally, we demonstrated that PLSCR1 is overexpressed in human breast cancer tissue and in human breast cancer cell lines especially in triple-negative MDA-MB-231 and MDA-MB-468. We also showed that knockout of PLSCR1 inhibits migration, invasion and anchorage independent growth of MDA-MB-231, and that TGFβ influences PLSCR1 cellular distribution in those cells.

Citation Format: Margaret Klauzinska, Hideaki Karasawa, Maria Cristina Rangel, Nadia Castro, Alyson Baker, Daniel Bertolette, Julie Krask, David Salomon. PLSCR1- a modifier of TGFβ 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 2226. doi:10.1158/1538-7445.AM2015-2226
Abstract 2231: Type II transglutaminase is an epidermal squamous cell carcinoma stem cell survival protein
Authors: Fisher Matt , Xu Wen , Adhikary Gautam , Kerr Candace , Eckert Richard
Published: August 2015

Cancer stem cells are thought to be responsible for rapid tumor growth, metastasis and enhanced tumor survival following drug treatment. For this reason, there is a major emphasis on identifying proteins that can be targeted to kill cancer stem cells or control their growth. Our studies suggest that transglutaminase type II (TG2) is such a target in epidermal squamous cell carcinoma. TG2 was originally described as a transamidase that catalyzes formation of ϵ-(γ-glutamyl)lysine bonds. However, subsequent studies reveal that TG2 is a GTP binding protein that plays an important role in cell signaling and survival. In the present study we show that TG2 is a target for anti-stem cell therapy in human squamous cell carcinoma. We identify TG2 as highly elevated in epidermal cancer stem cells (ECS cells) and show that TG2 knockdown or suppression of TG2 function with inhibitors reduces ECS cell survival, spheroid formation, matrigel invasion and migration. The reduction in survival is associated with activation of apoptosis. Mechanistic studies, using TG2 mutants suggest that the GTP-binding activity is required for maintenance of ECS cell growth and survival. These studies suggest that TG2 has an important role in maintaining cancer stem cell survival, invasive and metastatic behavior, and is an important therapeutic target to reduce survival of cancer stem cells in epidermal squamous cell carcinoma.

Citation Format: Matt Fisher, Wen Xu, Gautam Adhikary, Candace Kerr, Richard Eckert. Type II transglutaminase is an epidermal squamous cell carcinoma stem cell survival protein. [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 2231. doi:10.1158/1538-7445.AM2015-2231
Abstract 2246: Progesterone generates cancer stem cells through membrane progesterone receptor-triggered signaling in basal-like human mammary cells
Authors: Vares Guillaume , Sai Sei , Wang Bing , Fujimori Akira , Nenoi Mitsuru , Nakajima Tetsuo
Published: August 2015

Among the four main subtypes of breast cancer, basal-like breast cancer (the predominant subtype of triple-negative breast cancer) is of particular concern, due to its high frequency, relative lack of effective therapies and poor prognosis. Ionizing radiation (for example as treatment for other cancers) and cumulative exposure to steroid hormones (as seen in postmenopausal women under hormone replacement therapy) are known risk factors for breast cancer. Because of their tumor-initiating properties, cancer stem cells (CSCs) were proposed to be responsible for relapse and metastasis. In MCF10A non-cancerous basal-like PR-negative cells, progesterone treatment and 1 Gy X-rays generated ALDH+ and CD44+/CD24- CSCs. Here, we report that progesterone activated the Pi3k/Akt pathway via membrane progesterone receptor (mPR). Inhibition of the Pi3k/Akt pathway counteracted the generation of CSCs by progesterone and irradiation. The stimulation of Pi3K/Akt by progesterone resulted in the inactivation of FOXO3 and a downregulation of miR-29 expression. Stabilization of miR-29 expression impeded the generation of CSCs, while its inhibition alone was sufficient to generate CSCs. This study provides a new mechanistic basis for progesterone and radiation-induced breast cancer risk. In addition, the elucidation of new pathways and miRNA regulations involved in CSC generation and maintenance may open the door to potential new strategies for basal cancer primary or adjuvant therapy, by blocking non-CSCs from replenishing the pool of CSCs and by targeting mechanisms responsible for CSC maintenance. Such CSC-focused therapies may be effective at preventing metastasis and relapse.

Citation Format: Guillaume Vares, Sei Sai, Bing Wang, Akira Fujimori, Mitsuru Nenoi, Tetsuo Nakajima. Progesterone generates cancer stem cells through membrane progesterone receptor-triggered signaling in basal-like human mammary 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 2246. doi:10.1158/1538-7445.AM2015-2246
Abstract 2253: Cadherin 17 plays an important role in breast cancer metastasis to bone marrow
Authors: Okada Tomoko , Furihata Mutsuo , Kurabayashi Atsushi , Imamura Toru
Published: August 2015

To understand the mechanism underlying bone marrow metastasis in breast cancer, we have established a highly bone marrow metastatic mouse breast cancer cell line, 4T1E/M3. While metastatic potential of 4T1E/M3 to bone was quite high (about 80%) when injected intravenously to mice, it was rather low (about 20%) when injected subcutaneously. Then using 4T1E/M3 cells, we carried out the in vitro and in vivo selection and established higher bone marrow metastatic cells, FP10SC2, which have a very high potential of metastases to lungs (100%) and spines (85%) when subcutaneously injected to mice. DNA microarray analysis revealed that the gene expression of cadherin17 was upregulated in FP10SC2 compared to the parental 4T1E/M3. When the expression of cadherin17 was suppressed by RNAi, the anchorage independent growth and migration activity was dramatically decreased. Moreover, metastatic activity of cadherin17(-)/FP10SC2 to spines was significantly reduced compared to control/FP10SC2. When cadherin 17 was overexpressed in 4T1E/M3 parental cells, the colony formation and migration activity was clearly augmented. Taken together, it was suggested that cadherin 17 plays an important role on breast cancer metastasis to bone marrow.

Citation Format: Tomoko Okada, Mutsuo Furihata, Atsushi Kurabayashi, Toru Imamura. Cadherin 17 plays an important role in breast cancer metastasis to bone marrow. [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 2253. doi:10.1158/1538-7445.AM2015-2253
Abstract 2259: Requirement for BUB1B in tumor progression of lung adenocarcinoma
Authors: Chen Honglin , Lee James , Kljavin Noelyn M. , Haley Benjamin , Daemen Anneleen , Liang Yuxin , Johnson Leisa
Published: August 2015

Lung adenocarcinoma is often discovered as metastatic disease with very poor prognosis. However, much remains unknown about the mechanisms of lung adenocarcinoma tumor progression. In this study we showed that knockdown of BUB1B, a critical mitotic checkpoint protein, significantly inhibited anchorage-independent growth of lung adenocarcinoma cell lines. In allograft and tail vein mouse model studies, BUB1B suppression inhibited primary tumor growth and reduced metastasis to the lung and lymph nodes, resulting in prolonged survival in both tumor prevention and tumor intervention settings. Mechanistic studies revealed that BUB1B knockdown sensitized cells to anoikis. The N-terminal region of BUB1B was required for its functions in both anchorage-independent growth and anoikis resistance, whereas the kinase domain was less critical. Overexpression of BUB1B is associated with disease progression and poor survival in human lung adenocarcinoma patients. Collectively, these data reveal a novel function for BUB1B in mediating anchorage-independent survival and growth, thereby facilitating lung adenocarcinoma dissemination during metastasis. Thus, targeting BUB1B could provide potential therapeutic benefit in suppressing metastasis and prolonging survival in lung adenocarcinoma patients.

Citation Format: Honglin Chen, James Lee, Noelyn M. Kljavin, Benjamin Haley, Anneleen Daemen, Yuxin Liang, Leisa Johnson. Requirement for BUB1B in tumor progression of lung adenocarcinoma. [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 2259. doi:10.1158/1538-7445.AM2015-2259
Abstract 2264: Dual effects of podoplanin on the regulation of cancer cell gene expression and platelet CLEC-2 signaling during cancer metastasis
Authors: Tseng Ching-Ping , Chang Yao-Wen , Hsieh Pei-Wen , Cheng Ju-Chien
Published: August 2015

Podoplanin (PDPN) is frequently up-regulated in squamous cell carcinoma and is a marker for poor prognosis. PDPN facilitates cancer metastasis and tumor cell-induced platelet aggregation (TCIPA) by modulating cancer cell cytoskeleton reorganization and platelet C-type lectin-like receptor 2 (CLEC-2) activation. In this study, the effects of PDPN on the regulation of cancer cell gene expression and platelet CLEC-2 signaling are investigated. Using xenograft injection of the C6 glioma cells into nude mice as a metastatic model, the cells that colonized at lung tissue were isolated to establish a subline C6-Lung that was found to express high levels of PDPN. Knockdown of PDPN expression in C6-Lung resulted in the abrogation of TCIPA, indicating that PDPN plays a key role in platelet activation and TCIPA. Microarray analyses revealed differential gene expression signatures between low and high PDPN-expressed cells. Several genes were up-regulated (Sncg, Timp1, Mmp3 and Pla1a) and down-regulated (Calcb, Filip1, Agrn and Itga4) significantly in high PDPN-expressed C6-Lung cells. On the other hand, distinct platelet protein tyrosine phosphorylation profiles were revealed when platelets were treated with different CLEC-2 agonists including PDPN, aggretin and fucoidan. In addition to the activation of common CLEC-2 signaling proteins such as Src family kinase, Syk, and phospholipase Cγ2, Akt1/PDK1 and PKCμ were identified as the two novel signaling pathways activated by PDPN, indicating the presence of SFK-independent pathway downstream of CLEC-2. Our data together provide a definitive evidence for the presence of a unique network for PDPN-regulated cancer cell gene expression and platelet CLEC-2 signaling that shed a new insight for the functional roles of PDPN in cancer metastasis.

Citation Format: Ching-Ping Tseng, Yao-Wen Chang, Pei-Wen Hsieh, Ju-Chien Cheng. Dual effects of podoplanin on the regulation of cancer cell gene expression and platelet CLEC-2 signaling during cancer metastasis. [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 2264. doi:10.1158/1538-7445.AM2015-2264
Abstract 2269: A novel molecular subset of metastatic lung adenocarcinoma is defined by the function of the proteoglycan receptor HMMR
Authors: Stevens Laura E. , Zhao Minghui , Liu Zongzhi , Nguyen Don
Published: August 2015

Lung adenocarcinoma (LUAD) is a heterogeneous and deadly subtype of non-small cell lung cancer that metastasizes rapidly to distant organs. Recent transcriptional profiling of primary LUADs by TCGA identified three molecular subgroups with unique clinical and biological features. Amongst the most aggressive LUADs are tumors classified in the proximal inflammatory (PI) subtype. However, the molecular mechanisms that dictate metastatic competence of this tumor type remain undefined. Using a bioinformatic approach, we found that PI tumors are significantly enriched for the expression of extracellular matrix genes and their corresponding cell surface receptors compared to tumors of other subtypes. Among these receptors, hyaluronan mediated motility receptor (HMMR) was found to be significantly upregulated. Knockdown of HMMR in LUAD cells decreased their potential to invade, migrate, and form colonies in vitro. In an experimental mouse model of metastasis, suppression of HMMR decreased metastatic incidence and burden. Interestingly, using an orthotopic model of LUAD, suppression of HMMR attenuated tumor initiating capacity of LUAD cells within an inflammatory microenvironment without affecting tumor progression. In distant organs, HMMR is similarly essential for tumor re-initiation and expansion of micrometastases to macrometastases. Our findings suggest that the aggressive behavior of PI LUADs requires the interaction between HMMR and the tumor microenvironment.

Citation Format: Laura E. Stevens, Minghui Zhao, Zongzhi Liu, Don Nguyen. A novel molecular subset of metastatic lung adenocarcinoma is defined by the function of the proteoglycan receptor HMMR. [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 2269. doi:10.1158/1538-7445.AM2015-2269