bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1944: NFκB: Activation by EGF requires TLR4 and activation by LPS requires EGFR
Authors: De Sarmishtha , Zhou Hao , Li Xiaoxia , Stark George
Published: August 2015

In normal cells, quiescent NFκB is activated by inflammatory stimuli, whereas the abnormal constitutive activation of NFκB is important for the survival of most cancer cells. NFκB activation is regulated by several different pathways. Recently we showed that EGFR is an important mediator of NFκB activation, in both non-malignant and malignant cells. We now provide additional mechanistic insight into this pathway by further investigating whether signaling to NFκB in response to EGF depends solely on EGFR, or involves another receptor known to activate NFκB. Knockdown experiments revealed that TLR4 and two downstream components in the canonical TLR4 pathway, MYD88 and TAK1, are all required for EGF-dependent NFκB activation. Conversely, the ability of TLR4 to activate NFκB in response to LPS was impaired by down regulating EGFR expression or by use of the EGFR inhibitor erlotinib. PP2, a general inhibitor of SRC-family kinases, blocked EGF-induced NFκB activation, and the SRC family member LYN became bound to EGFR upon LPS stimulation. Erlotinib blocked this LPS-dependent association, indicating a key role for LYN in EGFR-TLR4 cross signaling. In mice, erlotinib blocked the LPS-induced, NFκB-dependent expression of TNFα and IL-6, indicating that EGFR is essential for LPS-induced NFκB activation in vivo.

Citation Format: Sarmishtha De, Hao Zhou, Xiaoxia Li, George Stark. NFκB: Activation by EGF requires TLR4 and activation by LPS requires EGFR. [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 1944. doi:10.1158/1538-7445.AM2015-1944
Abstract 1959: 15-lipoxygenase-1 suppression of colitis-associated colon cancer through inhibition of the IL-6/STAT3 signaling pathway
Authors: Zuo Xiangsheng , Mao Fei , Xu Min , Xu Weiguo , Tian Rui , Micheline J. Moussalli Micheline J. , Elias Elias , Li Haiyan S. , Watowich Stephanie S. , Shureiqi Imad
Published: August 2015

The IL-6/STAT3 pathway is a critical signaling pathway for colitis-associated colorectal cancer (CAC). PPAR-delta (PPAR-d), a lipid nuclear receptor, upregulates IL-6. 15-Lipoxygenase-1 (15-LOX-1) is crucial to produce lipid signaling mediators to terminate inflammation. 15-LOX-1 downregulates PPAR-d. 15-LOX-1 effects on IL-6/STAT3 signaling and CAC tumorigenesis are unknown. We here report that intestinally targeted transgenic 15-LOX-1 expression in mice (15-LOX-1-Gut mice) inhibited azoxymethane and dextran sodium sulfate-induced CAC, IL-6 expression, STAT3 phosphorylation, and IL-6/STAT3 downstream target expression (Notch and MUC1). 15-LOX-1 downregulation was associated with IL-6 upregulation in human colon cancer mucosa. Re-expression of 15-LOX-1 in human colon cancer cells suppressed IL-6 mRNA expression, STAT3 phosphorylation, IL-6 promoter activity, and PPAR-d mRNA and protein expression. PPAR-d overexpression in colonic epithelial cells (PPAR-d-Gut mice) promoted CAC tumorigenesis in mice and increased IL-6 expression and STAT3 phosphorylation, whereas concomitant 15-LOX-1 expression in colonic epithelial cells (15-LOX-1-PPAR-d-Gut mice) suppressed these effects [e.g., tumor number per mouse (mean ± SE) was 4.22 ± 0.68 in wild-type littermates, 6.67 ± 0.83 in PPAR-d-Gut mice (p = 0.026), and 2.25 ± 0.25 in 15-LOX-1-PPAR-d-Gut mice (p = 0.0006)]. Identification of 15-LOX-1 suppression of PPAR-d to inhibit IL-6/STAT3 signaling-driven CAC tumorigenesis provides mechanistic insights that can be utilized to molecularly target CAC.

Citation Format: Xiangsheng Zuo, Fei Mao, Min Xu, Weiguo Xu, Rui Tian, Micheline J. Micheline J. Moussalli, Elias Elias, Haiyan S. Li, Stephanie S. Watowich, Imad Shureiqi. 15-lipoxygenase-1 suppression of colitis-associated colon cancer through inhibition of the IL-6/STAT3 signaling 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 1959. doi:10.1158/1538-7445.AM2015-1959
Abstract 1977: Elucidating heterogeneity within cancer cell populations using single cell RNA detection
Authors: Weldon Don , Williams Yuko , Koong Victor , Ko Alex
Published: August 2015

As researchers continue to analyze the relationships between gene expression and biological processes involved in the development and progression of cancer, increasing importance is being placed on the study of genetic variation across single cells. However, traditional methods tend to measure RNA expression globally, with cell lysis and fixation steps resulting in a loss of key information about individual cells, making it challenging to properly assay the genetic heterogeneity within cell populations. We demonstrate the use of a single cell RNA detection technology that specifically measures gene expression in live cells without altering their viability or inherent biology. This enables researchers to not only detect heterogeneity within their samples, but also separate, enrich, and further characterize cell subpopulations based on RNA expression variation, when using techniques such as Fluorescent Activated Cell Sorting (FACS).

Here, we describe live cell detection of cancer biomarkers within heterogeneous cell models. First, we co-cultured MCF-7 and SK-BR3 breast cancer cell lines in specific ratios to mimic heterogeneous breast cancer populations. Subsequent characterization of the populations was performed by both flow cytometry using fluorescent live-cell RNA detection probes, and quantitative real-time PCR. Next, we analyzed miR-221 expression in a mixture of T47D and MDA-MB-231 breast cancer cell lines, which allowed us to sort the two cell types based on differential expression of this microRNA and perform downstream immunohistochemical analysis of vimentin and E-cadherin, two protein markers known to be associated with the epithelial-to-mesenchymal transition during tumor progression.

Use of a live cell RNA detection methodology in gene expression experiments allows scientists to formulate novel questions and design experiments that better address the importance of gene expression heterogeneity in normal and disease states, particularly in cancer, where individual cells within the tumor microenvironment may exhibit aberrant RNA expression profiles that render them invasive or metastatic.

Note: This abstract was not presented at the meeting.

Citation Format: Don Weldon, Yuko Williams, Victor Koong, Alex Ko. Elucidating heterogeneity within cancer cell populations using single cell RNA detection. [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 1977. doi:10.1158/1538-7445.AM2015-1977
Abstract 1987: Molecular characterization of the breast cancer-associated antigen NY-BR-1
Authors: Bitzer Julia , Kosaloglu Zeynep , Halama Niels , Ziegelmeier Claudia , Lerchl Tina , Spille Anna , Pudenz Maria , Koellensperger Eva , Eichmueller Stefan , Osen Wolfram , Schneeweiss Andreas , Marmé Frederik , Zoernig Inka , Jaeger Dirk
Published: August 2015

Breast cancer is one of the most common malignancies with increasing incidence every year and a leading cause of death among women. Although early stage breast cancer can be effectively treated, there are limited numbers of treatment options available for patients with advanced and metastatic disease.

The breast cancer associated antigen NY-BR-1 was identified by a serological screening strategy (SEREX). NY-BR-1 is expressed in the majority (>70%) of breast tumors as well as metastases, in normal breast tissue, in testis, and occasionally in prostate tissue. NY-BR-1 was confirmed as an immunogenic antigen as we could detect NY-BR-1 specific spontaneous humoral and cellular immune responses in several breast cancer patients.

However, biological function, regulatory mechanisms, and interaction partners of NY-BR-1 are still unknown. Therefore, we combined integrative functional analysis (genomics, transcriptomics and epigenetics) and experiments to unravel the transcriptional regulation of NY-BR-1 and to detect its function within the mammary gland.

As numerous binding sites for nuclear hormone receptors (e.g. Estrogen Receptor alpha, Progesterone receptor and Glucocorticoid receptor) and CpG islands were predicted within the promoter sequence of NY-BR-1, primary-tissues from breast reductions or tumor cells obtained from pleural effusions were treated with different hormones (Estradiol, Dexamethasone, Progesterone, Retinoic acid, Vitamin D) or with the HDAC inhibitors Valproic acid, Trichostatin A and the demethylating agent 5´Aza-Deoxycytidine in order to analyze the transcriptional regulation of NY-BR-1.

Additionally, we analyzed the methylation status at various sites within the promoter region of NY-BR-1 using EpiTyper MassARRAY technology and publicly available resources such as the TCGA database. Here, we present preliminary results of our analysis and evaluate their implication for further investigation of NY-BR-1.

Citation Format: Julia Bitzer, Zeynep Kosaloglu, Niels Halama, Claudia Ziegelmeier, Tina Lerchl, Anna Spille, Maria Pudenz, Eva Koellensperger, Stefan Eichmueller, Wolfram Osen, Andreas Schneeweiss, Frederik Marmé, Inka Zoernig, Dirk Jaeger. Molecular characterization of the breast cancer-associated antigen NY-BR-1. [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 1987. doi:10.1158/1538-7445.AM2015-1987
Abstract 1998: A novel luminescent system for monitoring intracellular protein:protein interactions
Authors: Binkowski Brock , Dixon Andrew S. , Schwinn Marie K. , Hall Mary P. , Zimmerman Kris , Otto Paul , Lubben Thomas , Butler Braeden , Kirkland Thomas , Wood Monika G. , Encell Lance P. , Fan Frank , Wood Keith V.
Published: August 2015

Protein:protein interactions (PPIs) are essential to the cellular signal transduction pathways that contribute to cancer. Although numerous approaches exist to monitor protein:protein interactions in vitro, methods for intracellular detection have been more limited. We developed a system for the detection of PPIs based on NanoLuc luciferase. Proteins are fused to either an 18 kDa or 1 kDa subunit. PPI facilitates interaction of the subunits, leading to increased luminescence. Intracellular PPIs are measured using a non-lytic assay protocol by providing a cell permeable substrate. The interaction status can be monitored at a single time point or followed continuously over 1-2 hours. Unlike related approaches where a luciferase enzyme is simply split, the subunits of this system were optimized for structural stability and minimal binding affinity. The result is a luminescence signal that is orders of magnitude brighter than approaches using split luciferase, allowing use of fusion protein expression levels at or near physiological levels. The low affinity interaction between subunits (Kd ∼200 μM) minimizes assay background by preventing non-specific association, and the system shows rapid reversibility using model systems such as protein kinase A catalytic and regulatory subunits. We have successfully applied this system to several PPIs related to cancer research, including TP53:MDM2, MYC:MAX, and members of the RAS-RAF signaling pathway.

Citation Format: Brock Binkowski, Andrew S. Dixon, Marie K. Schwinn, Mary P. Hall, Kris Zimmerman, Paul Otto, Thomas Lubben, Braeden Butler, Thomas Kirkland, Monika G. Wood, Lance P. Encell, Frank Fan, Keith V. Wood. A novel luminescent system for monitoring intracellular protein:protein interactions. [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 1998. doi:10.1158/1538-7445.AM2015-1998
Abstract 2012: Accurate measurement of free and complexed PSA concentrations in serum of women using a novel technology with fg/mL sensitivity
Authors: Nikolenko Galina N. , Stengelin Martin K. , Sardesai Laukik , Glezer Eli N. , Wohlstadter Jacob N.
Published: August 2015

Measurement of free and complexed PSA (prostate specific antigen; complexed with ACT) in certain patient groups such as prostatectomy patients and women at risk of breast and ovarian cancer has been limited by the sensitivity of current immunoassay technology (5-30 pg/mL for common clinical analyzers). Fifth generation assays for total PSA with detection limits of 0.05 to 0.1 pg/mL have been described (McDermet 2009; Thaxton 2009; Wilson 2011). We developed a next-generation assay format based on MSD's MULTI-ARRAY® electrochemiluminescence technology with detection limits of 2 fg/mL (0.002 pg/mL) for complexed PSA (cPSA) and 20 fg/mL for free PSA (fPSA), requiring only 25 μL of serum or plasma per measurement.

Assays were calibrated against the WHO standards 96/668 (free PSA) and 98/670 (90% complexed, 10% free PSA). The dynamic range of both assays was three to four orders of magnitude. Typical intra-plate coefficients of variation ranged from 5% to 15%. LLOQ's of cPSA and fPSA assays were 5 fg/mL and 55 fg/mL, respectively. Spike recovery and dilution linearity were between 80% and 120%. Approximately 100 serum samples from women were evaluated. Free PSA was detectable in 95% of samples and cPSA was detectable in all samples.

Multiple literature reports indicate that free PSA might be a marker for early detection of breast cancer; however, the concentrations of many samples were close to or below the detection limits of the assays used for the studies. We tested 48 serum samples from patients with breast cancer and 37 serum samples from apparently healthy women. As the table below shows, there was no clear difference between PSA concentrations in cases and controls.

Free PSA [fg/mL] (Median, IQR)cPSA [fg/mL] (Median, IQR)Free/cPSA (Median, IQR)Apparently Healthy (n = 37)150 (80-260)560 (210-1,500)0.29 (0.18-0.73)Breast Cancer (n = 48)90 (50-260)180 (90-480)0.36 (0.17-0.77)Breast Cancer, ER positive (n = 10)160 (80-660)170 (120-13,000)0.62 (0.22-1.8)Breast Cancer, HER2 positive (n = 10)160 (50-290)460 (110-8,300)0.10 (0.03-0.44)Breast Cancer, triple negative (n = 10)90 (40-370)140 (50-300)0.68 (0.34-1.9)

In conclusion, we developed a next-generation assay format that is 100 to 1000 times more sensitive than the currently available clinical PSA assays. This enables accurate determination of free PSA and cPSA concentrations in the serum of females.

Citation Format: Galina N. Nikolenko, Martin K. Stengelin, Laukik Sardesai, Eli N. Glezer, Jacob N. Wohlstadter. Accurate measurement of free and complexed PSA concentrations in serum of women using a novel technology with fg/mL sensitivity. [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 2012. doi:10.1158/1538-7445.AM2015-2012
Abstract 2031: RRD-251 enhances retinoic acid induced differentiation of myeloblastic leukemic cells
Authors: Wallace Aaron S. , Geil Wendy M. , Yen Andrew
Published: August 2015

All-trans-retinoic acid (RA) is known to induce terminal granulocytic differentiation and cell cycle arrest in HL-60 cells. This is driven by sustained activation of the MAPK pathway, where Raf-1 (MAPKKK) acts as a critical signaling molecule. RRD-251 is a small molecule that binds to the Rb protein and prevents its interaction with Raf-1. Hyperphosphorylation of Rb leads to a conformational change, inhibiting its ability to sequester the E2F family of transcription factors. To determine the effect of RRD-251 on RA-induced differentiation, cells were treated with RA and/or RRD-251 and then analyzed for proliferation, expression of key markers of differentiation, cell cycle arrest, and modifications in protein levels. RRD-251 by itself significantly inhibited proliferation, but cells treated with both RA and RRD-251 showed even further reduction in cellular growth. Similarly, both RA and RRD-251 induce cell cycle arrest, but cells treated with both show higher levels of G0/G1 than singly treated cells. Expression of CD11b is used a late classical marker for differentiation in HL-60. RRD-251 does not induce expression of CD11b, but cultures treated with RRD-251 and RA have nearly double the amount of cells expressing CD11b as compared to RA treated cells alone 72 hours after treatment. Additionally, we had wanted to see the relationship between specific Rb phosphorylation, the cell cycle, and treatment of RA and RRD-251. At 72 hours after treatment, RA induces the formation of a population of cells with no phosphorylation at serine 608 of Rb, which was 99.9% in G0/G1. The appearance of two populations for Rb phosphorylation was unique to serine 608 out of the four phospho-sites that we studied: serine 608, serine 780, serine 795, serine 807/811. Cells treated with both RA and RRD-251 had more cells in the no-phosphorylation population at 72 hours, as well as the appearance of this population at 48 hours. Finally, we observed the changes in protein amounts and localizations by western blotting. RA is known to cause the phosphorylation of Raf-1 at serine 621 and its translocation to the nucleus at 48 hours. This was observed on our blots and that cells treated with both RA and RRD-251 had increased amounts of total nuclear Raf-1 and of pS621 Raf-1 at 48 and 72 hours. The results reported here further support the usefulness of RRD-251 as an anti-cancer drug, from reducing tumor size and preventing angiogenesis in drug-resistant melanoma, to enhancing retinoic acid-induced differentiation in myeloblastic leukemic cells.

Citation Format: Aaron S. Wallace, Wendy M. Geil, Andrew Yen. RRD-251 enhances retinoic acid induced differentiation of myeloblastic leukemic 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 2031. doi:10.1158/1538-7445.AM2015-2031
Abstract 2038: A novel role for the NLRP3 inflammasome in lung cancer
Authors: Petrilli Virginie , Bodnar Mélanie , Guey Baptiste , Hacot Sabine , Lantuejoul Sylvie
Published: August 2015

Although significant advances have been made in the diagnosis and treatment of lung cancer, it remains the most fatal cancer worldwide. This poor outcome is mainly due to the limited curative options available especially for advanced cancer stages. About 80% of lung cancers are Non Small Cell Lung Cancers (NSCLC). Although several mutations in oncogenes and tumor genes have been identified in NSCLC, the oncogene K-ras and the Receptor Tyrosine Kinases (RTK) being the most frequently mutated, about 40% of cancers are driven by unidentified mutations. Many cancer cells activate a proinflammatory program to manipulate the host immune system to support their growth. Inflammasomes are crucial effectors of the innate immunity. Upon activation, inflammasomes assemble into multiprotein complexes controlling the activation of the inflammatory caspase-1 and the maturation of the pro-Interleukin-1beta (pro-IL-1b) and the pro-IL-18 into active cytokines. The NLRP3 inflammasome detects pathogens, airborne pollutants but also self-danger signals released by dying cells. In the lung, hyperactivation of this complex upon bleomycin or asbestos exposure drives an inflammatory response resulting in lung fibrosis.

Published large-scale genomic analyses of NSCLC identified somatic mutations in the NLRP3 gene and scored NLRP3 as a putative oncogene. Little is known about the role of the NLRP3 inflammasome in lung cancer. We speculated that the NLRP3 inflammasome could play a prominent role in NSCLC and studied the function of NLRP3 inflammasome in human non-transformed and transformed lung epithelial cells. Our data unravel new functions for the NLRP3 inflammasome in DNA damage sensing and unexpectedly suggest that NLRP3 could act as a tumor suppressor gene in lung cancer.

Citation Format: Virginie Petrilli, Mélanie Bodnar, Baptiste Guey, Sabine Hacot, Sylvie Lantuejoul. A novel role for the NLRP3 inflammasome in lung 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 2038. doi:10.1158/1538-7445.AM2015-2038
Abstract 2052: The transcriptional co-regulator and emerging cancer drug target C-terminal binding protein (CtBP) is a transforming oncogene
Authors: Sumner Evan T. , Grossman Steven R.
Published: August 2015

The paralogous C-terminal binding proteins (collectively CtBP) 1 and 2 are evolutionarily conserved transcriptional co-regulators which demonstrate oncogene-like properties such as allowing for the progression of EMT and cellular invasion/migration, and supporting cellular survival through suppression of tumor suppressors (PTEN, p16, E-cadherin, BRCA-1) and pro-apoptotic genes (Bik, Puma, Noxa). Overexpression of CtBP is found in colon, ovarian, breast, and prostate cancers, and often correlates with loss of the tumor suppressors ARF and APC and with poorer prognosis for the patient. CtBP is also an emerging cancer drug target, encoding a dehydrogenase domain that can be targeted with small molecule inhibitors to kill cancer cells. CtBP appears to fit the criteria for a driver oncogene, though has never been fully characterized as such. To investigate the hypothesis that CtBP is a transforming oncogene, we subjected CtBP2 to multiple assays for cellular oncogenic activity including loss of contact inhibition, invasion, and anchorage independent growth. Previously immortalized NIH 3T3 mouse fibroblasts expressing CtBP2 or mutant H-Ras as a positive control were analyzed for focus formation, Matrigel-invasion, and soft agar colony formation. Consistent with the hypothesis that CtBP exhibits properties of an oncogene, CtBP2 potently induced focus formation, Matrigel-invasion, and soft agar colony formation with similar potency to oncogenic mutant H-Ras. Moreover, CtBP2 cooperated with the adenovirus E1A oncoprotein but not H-Ras to transform primary mouse embryonic fibroblasts (MEFs). Taken together, the 3T3 and MEF transformation data suggest CtBP2 acts as a “Ras-like” oncogene in rodent cell transformation assays. Previous research from our lab has shown that CtBP's ability to induce migration in HCT116 cells depends in part on its activation of T-cell lymphoma invasion and metastasis 1 (Tiam1). Work is ongoing to characterize critical downstream targets for CtBP's transforming activity. Targeting CtBP and its dehydrogenase domain in cancers where the protein is found to be overexpressed, or where its regulators, ARF and APC are found to be inactive, could lead to a therapeutic approach specific for an emerging driver oncogene.

Citation Format: Evan T. Sumner, Steven R. Grossman. The transcriptional co-regulator and emerging cancer drug target C-terminal binding protein (CtBP) is a transforming oncogene. [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 2052. doi:10.1158/1538-7445.AM2015-2052
Abstract 2053: Overexpressed Rab1A is associated poor prognosis and promotes oncogenic growth and metastasis through mTORC1 activation in hepatocellular carcinoma
Authors: Xu Bi-Hong , Li Xiao-Xing , Wang Hui-yun , Zheng X.F. Steven
Published: August 2015

Background: The mammalian target of rapamycin complex 1 (mTORC1) is a pivotal regulator of cell growth and proliferation in response to nutrition and various factor. In recent years, studies have focus on over-activation of mTORC1 in normal cells leading to tumorigenesis and malignant tumor development. However, the precise factors of mTORC1 hyperactivation to promote the growth of hepatocellular carcinoma (HCC) or other cancers are still not well characterized. Thus, in this study, we aimed to clarify a novel gene -Rab1A, and it's expression and functional role in mTORC1 signal in of HCC. Methods: We analyzed the Rab1A protein in HCC and para-cancer samples from 143 patients and the correlation with its survival. To identify Rab1A's role in tumor progression, a serious of functional study were applied including overexpression and knockdown Rab1A in different cell lines both in vivo and in vitro. For mechanism research, western bolt was demonstrated to identify the signal dysregulation between Rab1A abnormity and mTORC1 pathway under involvement of irritants. Results: Our results showed that, compared with normal livers, Rab1A was frequently overexpressed in HCC and significantly associated with HCC prognosis. Functional study demonstrated that overexpression of Rab1A in HCC cells increased cell growth, cell migration, cell cycle progression and tumor formation in vivo and/or in vitro, all of which were effectively inhibited when Rab1A was silenced by shRNA. We further provided evidences that Rab1A promotes HCC growth and migration through phosphorylation of S6K, the consequence of activating mTORC1 signaling pathway. Mechanism research of the signal pathway between Rab1A and mTORC1 suggest that Rab1A is likely to stimulate mTORC1 directly, with the cooperation of Amino acid stimulation but not depend on the pathway of PI3K/AKT and MAPK/ERK..

Citation Format: Bi-Hong Xu, Xiao-Xing Li, Hui-yun Wang, X.F. Steven Zheng. Overexpressed Rab1A is associated poor prognosis and promotes oncogenic growth and metastasis through mTORC1 activation in hepatocellular carcinoma. [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 2053. doi:10.1158/1538-7445.AM2015-2053
Abstract 2055: Overexpression of nuclear met activates NF-κB signaling to promote tumorigenesis and metastasis in hepatocellular carcinoma
Authors: Tey Sze Keong , Tse Edith Yuk Ting , Ko Frankie Chi Fat , Yam Judy Wai Ping
Published: August 2015

Met is a surface receptor tyrosine kinase which upon hepatocyte growth factor (HGF) induction, triggers a diversity of downstream signaling cascades that modulates different cellular processes. Frequently, Met signaling pathway is hijacked in human cancers, resulted in abberant expression and activity. Emerging evidence has shown the presence of nuclear Met (nMet) in some cancerous tissues and cell lines, postulating that nMet could have unexplored functions within nucleus. Up to date, therapeutic antibodies and kinase inhibitos that undergoing clinical trial are all targeting Met surface receptor, which in this case exhibit limitation in acting against nMet. Althought Met is well known of its oncogenic role in hepatocellular carcinoma (HCC), existence and functions of nMet in HCC are yet to be reported. Our present study aim to examine the clinical association and functions of nMet in HCC. Immunohistochemistry of 103 human HCC paired samples showed that nMet was overexpressed in nearly 90% of cases. We also found that nMet overexpression was progressively increased along HCC development, from non-tumorous liver tissue to advanced HCC. Nonetheless, nMet overexpression was significantly associated with venous invasion and poorer overall survival in HCC patients. This suggests that nMet may play a pivotal role in HCC tumorigenesis and metastasis. As revealed by immunoblot and immunofluorescence, we found that nMet is about 48kDa and comprises of cytoplasmic domain of Met, as it can only be detected by an antibody against the carboxyl terminal of Met (C28). To study the functions of nMet, we employed a lentiviral based inducible exression system that tightly controlled by doxycycline to express the cytoplasmic region of Met. Immunofluorescence microscopy showed that juxtamembrane deletion of Met accumulates in the nucleus whereas truncated tyrosine kinase domain accumulates in the cytoplasm, implying a region necessary for nuclear localization of the cytoplasmic fragment. In vitro functional assay showed that nMet significantly promoted HCC cell proliferation and anchorage independent growth. It also significantly augmented HCC cell migration and invasiveness. Besides that, nMet also enhanced HCC tumor formation in animal model. Furthermore, we showed that nMet activates NF-κB reporter activity which might enhances the pro-metastatic gene expression to promote tumor invasiveness and aggressiveness.

Citation Format: Sze Keong Tey, Edith Yuk Ting Tse, Frankie Chi Fat Ko, Judy Wai Ping Yam. Overexpression of nuclear met activates NF-κB signaling to promote tumorigenesis and metastasis in hepatocellular carcinoma. [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 2055. doi:10.1158/1538-7445.AM2015-2055
Abstract 208: Novel intracellular Doppler imaging predicts therapeutic efficacy for personalized medicine
Authors: Turek John J. , Merrill Dan , Custead Michelle , Childress Michael , Nolte David D. , Yakubov Bakhtiyor , An Ran , Matei Daniela
Published: August 2015

This study reports first application of biodynamic imaging (BDI), a novel Doppler imaging technology, in studies of ovarian cancer xenografts and preclinical trials of canine multicentric lymphoma responding to doxorubicin.

In a study of A2780 human ovarian cell lines implanted in mice, an unexpected and large difference was discovered between mouse tumor explants versus A2780 cells grown as 3D tumor spheroids. The biopsied explants from mice were three times more sensitive to platinum, applied in vitro, than were multicellular tumor spheroids. A preclinical trial has been completed for canine non-Hodgkins lymphoma using BDI to predict patient outcome under doxorubicin therapy. The 3D imaging capability of BDI enabled the measurement of tumor heterogeneity in response to the therapy. The biodynamic assay took only 24 hours to complete, while the clinical outcome was often unknown for one to several months. The therapeutic efficacy of doxorubicin was predicted by BDI with 90% accuracy. Based on this success, biodynamic imaging is entering human pilot trials in ovarian, pancreatic, and esophageal cancer.

Biodynamic imaging is a radically different approach to the prediction of therapeutic efficacy for personalized medicine with higher accuracy and more biological relevance than standard cell-based assays. Supported in part by NIH NIBIB 1R01EB016582-01.

Citation Format: John J. Turek, Dan Merrill, Michelle Custead, Michael Childress, David D. Nolte, Bakhtiyor Yakubov, Ran An, Daniela Matei. Novel intracellular Doppler imaging predicts therapeutic efficacy for personalized medicine. [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 208. doi:10.1158/1538-7445.AM2015-208
Abstract 2088: PU.1 enrichment to intronic enhancers is required for meis1 gene expression in both hematopoiesis and leukemogenesis
Authors: Li Yajun , Li Bo , Li Qiang , Zhang Lixia , Li Ping
Published: August 2015

Hematopoiesis is a tightly regulated process with multiple transcription factors (TFs) involved sequentially. Aberrant expression of the lineage specific transcription factors may disturb normal differentiation of hematopoietic cells and result in leukemia. Myeloid Ectropic viral Integration Site 1 (MEIS1), induces and maintains MLL transformation, and plays a rate-limiting regulatory role in MLL leukemogenesis. Meis1 gene is identified as the downstream targets of PU.1, another important hematopoietic transcription factor, which is inversely correlated with worse prognosis. Though multiple regulatory elements have been identified throughout the Meis1 locus, little is known about how PU.1 plays its role on Meis1 gene expression.

To gain insight into the transcriptional regulatory role of PU.1 during myeloid differentiation, we isolated the hematopoietic stem/progenitor cells (HSPC, Lin-kit+CD34+ cells) and matured myeloid cells (Lin+Gr1+cells) from bone marrows of C57 mice, performed ChIP sequencing assay, and analyzed PU.1 enrichment and histone modifications. The ChIP seq data was then confirmed with an induced differentiation model of mouse MLL-ENL leukemia cells. In addition to DNaseI hypersensitivity, the PU.1 binding elements were also characterized with H3K4me1 and H3K27ac modification. We studied the transcriptional activation role of one common PU.1 binding elements with luciferase assay. Insertion of PU.1 binding elements before the promoter increased the luciferase reporter activity, suggesting that the PU.1 binding region be the enhancer element of Meis1 gene. The association between PU.1 enrichment and Meis1 gene expression was studied in further with PU.1 knocking down assay in the MLL leukemia cells. Bioinformatics analysis results suggest that there were other transcription factors, including HOXA9 and MEIS1 itself enriched around the PU.1 binding sites. Our results suggest that PU.1 enrichment to the intronic enhancers of Meis1 gene is required for the proper expression during hematopoiesis. PU.1 together with histone modification and many other TFs, including MEIS1 itself, contributed to the hematopoietic expression of Meis1 gene. (The work is supported by National Natural Science Foundation of China (Grant number 81100380) to Y.L.)

Citation Format: Yajun Li, Bo Li, Qiang Li, Lixia Zhang, Ping Li. PU.1 enrichment to intronic enhancers is required for meis1 gene expression in both hematopoiesis and leukemogenesis. [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 2088. doi:10.1158/1538-7445.AM2015-2088
Abstract 209: A quantitative analysis of paclitaxel-induced mitotic catastrophe of MDA-MB-231 cells by high frequency ultrasound
Authors: Pasternak Maurice M. , Czarnota Gregory J.
Published: August 2015

In recent years, high-frequency ultrasound (HFUS) has shown promise of non-invasively detecting tumor death in response to chemotherapy. However, those studies have been limited to ultrasound B-mode and immunohistochemical image comparison over extended periods of time (≥24 hours). The purpose of this study was to evaluate the relationship of quantified HFUS spectral parameters to the dynamics of tumor cell death in MDA-MB-231 cell samples exposed to paclitaxel chemotherapy. Several ultrasound parameters were investigated, including the, attenuation, spectral slope, spectral y-intercept, midband fit, estimated scatterer diameter and estimated scatterer concentration. Changes in these spectral-based parameters were evaluated in their capacity to respond to various cell death events, including mitochondrial depolarization, caspase activation, phosphatidylserine exposition, and membrane selective permeability compromise, as determined by flow cytometry analysis. Standard histology and transmission electron microscopy (TEM) permitted further investigation of possible candidates responsible for the changes detected using ultrasound. Based on TEM, negative TUNEL staining, and cell cycle analysis it was found that paclitaxel at 1mM induces mitotic catastrophe as the main cell death modality. The spectral slope correlated strongly (r2 = 0.89) with the percentage of cells arrested at the G2/M checkpoint and exhibited an opposite trend to that of systems undergoing classical apoptosis, suggesting that it may be a prime biophysical marker for differentiating modalities of tumor cell death. Midband fit increases through time suggested an increase in the concentration of acoustic scatterers. The formation of condensed chromatin condensates of a 1 micron or greater diameter in the course of paclitaxel-induced mitotic catastrophe, as revealed by TEM, were hypothesized to act as large intracellular scatterers and correlated with the increase in ultrasound midband fit. Midband fit showed the strongest correlation of all parameters (r2 = 0.99) when plotted against the percentage of the cell population expected to possess these scattering structures, establishing the first quantitative correlation of ultrasound parameters to a cell death index. These findings support that HFUS spectral parameters may be used to quantify the degree of cell death and predict the efficacy of chemotherapeutic treatment within a short post-treatment timeframe.

Citation Format: Maurice M. Pasternak, Gregory J. Czarnota. A quantitative analysis of paclitaxel-induced mitotic catastrophe of MDA-MB-231 cells by high frequency ultrasound. [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 209. doi:10.1158/1538-7445.AM2015-209
Abstract 2091: IL-8 mRNA gene expression up-regulated level in both primary culture cell and tissue biopsy from gastric cancer patient without Helicobacter pylori infection and down- regulated level predict clinical response after chemo
Authors: Limpakan (Yamada) Sirikan , Chakrabandhu ML.Bandhuphat , Kato Shunji , Wongsirisin Pattama
Published: August 2015

Background: There is some recent in vitro study reported that IL-8 mRNA gene had over expression when Helicobacter pylori was co- infected. In the Northern Thai population, we found independently IL-8 mRNA gene expression significantly rising level in gastric tissue biopsies when comparing to AGS cell line, and mainly in non-cag A H. pylori infection or without H. pylori infection.

Aim: The author aim to demonstrate the IL-8 mRNA gene expression level in vitro and in vivo study in the same host in order to see the change of its level in primary culture cell in same host gastric cancer patients, tissue biopsy, and AGS cell line. The comparative level before and after chemotherapy treatment were targeted for clinical response prediction.

Material and Methods: The control background of corpus normal epithelial and gastric adenocarcinoma biopsy tissues were treated for primary cell culture with single cell isolation technique. The histopathology, background atrophy, and H. pylori infection was confirmed. Biopsy tissues from tumor site also were collected at the same time before and after chemotherapy treatment in patient who received neo-adjuvant chemotherapy by FOLFOX IV regimen due to locally advance stage and unresectable tumor. The IL-8 mRNA gene expression was tested by relative quantitation real time RT-PCR.

Results: IL-8 mRNA gene expression can be significantly higher up-regulated level to 1.5-2.2 folds in gastric cancer tissue biopsy comparing to from 0.35-1.16 fold of primary culture cell without H. pylori infection when comparing to normal background of gastric epithelial tissue, and AGS cell line. The IL-8 mRNA gene expression down-regulated level is also remark decreased and/or undetected after chemotherapy treatment in correlation with patients who had well clinical response as a preliminary result.

Conclusion: We first demonstrated that the IL-8 mRNA gene expression is detected in primary gastric adenocarcinoma cell and its up-regulated level in tissue biopsies independently related to H. pylori infection in the same gastric cancer host background. The pilot sample size is needed to confirm in further clinical trial on chemotherapy as a new biomarker for advance gastric cancer treatment.

Citation Format: Sirikan Limpakan (Yamada), ML.Bandhuphat Chakrabandhu, Shunji Kato, Pattama Wongsirisin. IL-8 mRNA gene expression up-regulated level in both primary culture cell and tissue biopsy from gastric cancer patient without Helicobacter pylori infection and down- regulated level predict clinical response after chemo [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 2091. doi:10.1158/1538-7445.AM2015-2091
Abstract 2093: Regulation of c-Myc expression by aspirin and salicylic acid in colon cancer cells through a novel pathway
Authors: Ai Guoqiang , Dachineni Rakesh , Muley Pratik , Tummala Hemachand , Gunaje Jayarama B.
Published: August 2015

Epidemiological studies have demonstrated a significant correlation between regular aspirin use and reduced colon cancer incidence and mortality; however, the exact pathway has not been identified. We hypothesized that aspirin's anti-cancer effect may occur through modulation of c-Myc gene expression. Being a transcription factor, c-Myc controls nearly 15% of the expression of all genes and its activity is highly regulated in normal cells. In many cancers including colon cancer, c-Myc is mutated and constitutively activated which leads to uncontrolled cell proliferation. In the present study, we demonstrate that aspirin and its primary metabolite, salicylic acid, significantly decrease the c-Myc protein levels in colon cancer cells. In total cell lysates, both drugs decreased the levels of c-Myc in a concentration dependent (250 μM to 2500 μM) fashion (up to 70%). Interestingly, greater inhibition was observed in the nucleus than the cytoplasm, and immuno-fluorescence studies further confirmed these observations. Pre-treatment of cells with lactacystin partially reversed the inhibitory effect of both aspirin and salicylic acid, suggesting that 26S proteasomal pathway is involved. Transfection of HCT-116 cells with DDK-tagged c-Myc produced abundant c-Myc protein; but, it was unaffected by aspirin and salicylic acid. In the same lysates, the endogenous c-Myc level were significantly decreased. HPLC analysis showed that aspirin taken up by cells was metabolized to salicylic acid in a time-dependent fashion. This further supports the finding aspirin's inhibitory effect on c-Myc occurs through formation of salicylic acid. Our result suggests that aspirin and salicylic acid regulate c-Myc level at both transcriptional and post-transcription level. Inhibition of c-Myc levels may represent an important pathway by which aspirin exerts its anti-cancer effect and decrease the occurrence of cancer in epithelial tissues.

Citation Format: Guoqiang Ai, Rakesh Dachineni, Pratik Muley, Hemachand Tummala, Jayarama B. Gunaje. Regulation of c-Myc expression by aspirin and salicylic acid in colon cancer cells through a novel 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 2093. doi:10.1158/1538-7445.AM2015-2093
Abstract 2094: GSK-3 is a novel target of CREB and GSK-3-CREB signaling participates in cell survival in lung cancer
Authors: Park Sin-Aye , Lee Jong Woo , Herbst Roy S. , Koo Jaseok Peter
Published: August 2015

The transcription factor cyclic AMP-response element-binding protein (CREB) regulates diverse cellular processes, including cell differentiation, proliferation, survival, glucose metabolism, immune regulation, and synaptic plasticity associated with memory. Previously, we showed that CREB is critical on the regulation of mucous differentiation of normal human tracheobronchial epithelial (NHTBE) cells. In addition, the overexpression of CREB is associated with negative prognosis of patients with non-small cell lung cancer (NSCLC). Further studies showed that the knockdown of CREB suppresses the growth of lung cancer cells. It has been demonstrated that genes regulated by CREB have been reported to be involved in the suppression of apoptosis, induce cell proliferation, inflammation and tumor metastasis. However, the critical target genes of CREB in lung cancer have not been well understood. In this study, we identified GSK-3 as one of the CREB target genes critical for the growth and survival of lung cancer cells. The CREB knockdown significantly reduced the expression of GSK-3 and the direct binding of CREB on the promoter of GSK-3 was identified. Kaplan-Meier analysis with a public database showed a prognostic significance of aberrant GSK-3 expression in lung cancer. Inhibition of GSK-3 suppressed cell proliferation, colony formation and tumor growth. Although GSK-3 has been studied as a tumor suppressor in certain types of cancer, there is increasing evidence that GSK-3 plays an oncogenic role in various human cancers. Here, for the first time, we demonstrated that GSK-3 is regulated by CREB in lung cancer and is required for the survival of lung cancer cells. These findings implicate CREB-GSK-3 axis as a novel therapeutic target for lung cancer treatment.

Citation Format: Sin-Aye Park, Jong Woo Lee, Roy S. Herbst, Jaseok Peter Koo. GSK-3 is a novel target of CREB and GSK-3-CREB signaling participates in cell survival in lung 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 2094. doi:10.1158/1538-7445.AM2015-2094
Abstract 2097: Grainyhead-like 2 (GRHL2) regulates epithelial plasticity and stemness in oral cancers
Authors: Chen Wei , Mehrazarin Shebli , Shin Ki-Hyuk S , Lin Yi-Ling , Kim Reuben H. , Park No-Hee , Kang Mo K.
Published: August 2015

Purpose: Grainyhead-like 2 (GRHL2) is one of the three mammalian homologues of Drosophila Grainyhead involved in epithelial morphogenesis. GRHL2 also controls epithelial cell proliferation and differentiation. Our objective was to evaluate the role of GRHL2 in oral carcinogenesis and the underlying mechanism.

Experimental Design: GRHL2 expression was evaluated in multiple oral cancer cell lines and tissues of oral squamous cell carcinomas (OSCCs) by qRT-PCR, western blot assay, immunohistochemistry and laser-captured microdissection assay. The effects of shRNA-mediated GRHL2 knockdown were evaluated by tumor spheroid assay, colony formation assay, gene promoter luciferase assay and immunofluorescent staining and chromatin immunoprecipitation assay. In vivo the effect of GRHL2 loss on cancer cell tumorigenicity was evaluated by tumor xenograft assay.

Results: GRHL2 expression was elevated in cells and tissues of oral squamous cell carcinomas (OSCCs) compared with normal counterparts. Knockdown of GRHL2 resulted in the loss of in vivo tumorigenicity, cancer stemness, and epithelial phenotype of oral cancer cells. GRHL2 inhibition decreased oral cancer cell proliferation and colony formation. GRHL2 regulated the expression of miR-200 family and Octamer-binding transcription factor 4 (Oct-4) genes through direct promoter DNA binding. Overexpression of miR-200 genes in the oral cancer cells depleted of GRHL2 partially restored the epithelial phenotype, proliferative rate and cancer stemness.

Conclusion: GRHL2 plays pivotal role in the maintenance of the transformed phenotype in OSCCs. Downregulation of GRHL2 leads to the decrease of tumorigenicity, cancer stemness of oral cancer cell, in which miR-200 and Oct-4 genes partially mediate the tumorigenic effects of GRHL2 in OSCC. This study was supported in part by the grants R56DE024593 and Jack Weichman Endowed Fund.

Citation Format: Wei Chen, Shebli Mehrazarin, Ki-Hyuk S Shin, Yi-Ling Lin, Reuben H. Kim, No-Hee Park, Mo K. Kang. Grainyhead-like 2 (GRHL2) regulates epithelial plasticity and stemness in oral cancers. [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 2097. doi:10.1158/1538-7445.AM2015-2097