Factor Sp1 Research Articles

Chronic Myeloid Leukemia Blood Inflicted Injury in Cord Derived Wharton's Jelly Mesenchymal Stem Cells.

To determine the effects of blood from CML patients on human umbilical cord derived Wharton's jelly mesenchymal stem cells (WJMSCs) for evaluation of their therapeutic potential. An experimental study. Centre for Research in Molecular Medicine, University of Lahore, from September 2013 to December 2014. Possible behavior of WJMSCs in CMLpatients was assessed by culturing these cells in their plasma. WJMSCs at passage 3 were cultured in plasma isolated from 9 CMLpatients as well as 9 normal subjects. Effects on cell viability, proliferation, LDH release, paracrine factors (p38 and p53) and oxidative stress were evaluated. WJMSCs cultured in plasma of CML patients showed decreased viability, slow proliferation, high LDH release, high expression of p38 and p53 and a high oxidative stress compared to normal subjects. Stressed environment of CML patients' blood/plasma induced injury to WJMSCs as well as reduced their viability. Effectiveness of these cells for therapeutics of CML is, therefore, likely to be reduced.

Derepression of MicroRNA-138 Contributes to Loss of the Human Articular Chondrocyte Phenotype.

To investigate the function of microRNA-138 (miR-138) in human articular chondrocytes (HACs). The expression of miR-138 in intact cartilage and cultured chondrocytes and the effects of miR-138 overexpression on chondrocyte marker genes were investigated. Targets of miR-138 relevant to chondrocytes were identified and verified by overexpression of synthetic miRNA mimics and inhibitors, luciferase assays, chromatin immunoprecipitation, and RNA immunoprecipitation of native argonaute 2, using quantitative polymerase chain reaction, Western blotting, and luciferase assays. Expression levels of miR-138 were maintained at relatively low levels in intact human cartilage but were greatly increased upon loss of the differentiated phenotype in culture, with a concomitant decrease in the major cartilage extracellular matrix component COL2A1. We showed that miR-138 is able to repress the expression of COL2A1 by directly targeting Sp-1 and hypoxia-inducible factor 2α (HIF-2α), 2 transcription factors that are essential for COL2A1 transcription. We further demonstrated a direct association of these targets with miR-138 in the RNA-induced silencing complex and confirmed binding of Sp-1 to the COL2A1 promoter region in HACs. We propose that an evolutionary pressure helps to suppress expression levels of miR-138 in human cartilage, thus enabling expression of appropriate tissue-specific matrix genes. Inhibition of miR-138 may serve as a potential therapeutic strategy to maintain the chondrocyte phenotype or reduce the progression of dedifferentiation in cultured HACs.

Impact of the -216G/T Polymorphism on the Sp1 and Epidermal Growth Factor Receptor Interaction in Non-Small Cell Lung Carcinoma

The epidermal growth factor receptor is central to the growth, differentiation and the mobility of normal and cancer cells. Notably, EGFR plays an important role in non-small cell lung carcinoma development. Two known nucleotide variants in EGFR promoter at position -216 (G > T) and -191 (C > A) are known to influence promoter activity. The transcription factor Sp1 (Specificity protein 1) binds with higher affinity the T allele at position -216 which results in 30% increased transcriptional activity of EGFR. Sequencing of EGFR promoter region and exon 1 in 18 patients with pulmonary carcinoma revealed that -216 G/T variant was associated with 58.82% of NSCLC patients especially those with squamous carcinoma, with predominance of homozygote (T/T) variants. Strikingly, the -191C/A polymorphism was detected in 11.11% of patients having a pulmonary carcinoma with the predominance of homozygote (C/C) variants. The distribution analysis of the four haplotypes (G-C, G-A, T-C and T-A) and the diplotype (G-C/T-C) of -216G/T and -191C/A polymorphisms, revealed a clear predominance of T-C haplotype (216T-191C) in squamous cell carcinoma. The G-C and G-A haplotypes have a lesser distribution while the T-A haplotype is non-existent. The incidence of (G-C/T-C) diplotype is more important than the G-A haplotype in all the studied cases. Our results are strongly correlated with the data of Caucasian population.

Abstract A15: A human mini brain model of pediatric GBM

Abstract Glioblastoma (GBM) is the most devastating brain cancer with dismal prognosis both in adults and children and accounts for 16% of primary brain tumors. Despite aggressive therapeutic approaches, the majority of patients die within one year after diagnosis. Giving the limitation of current GBM mouse models, which do not reflect the biological properties of human tumors, in particular, tumor heterogeneity and pathogenesis, there is a growing need for new models. Current in vitro 3D models involve the use of hydrogel scaffolds and cell lines, which do not represent a viable developmental context where the tumor mass interacts with and invades the surrounding brain tissues. Starting with pluripotent human embryonic stem cells we reproduced a 3D tissue structure (so-called cerebral organoid) representing a developing human brain in vitro. We engineered these mini-brains to express oncogenic hits responsible for the derivation of tumors in pediatric GBM patients. Pediatric GBM is driven by a combination of epigenetic and genetic hits including the expression of a mutant histone H3 variant H3F3A (mutated at K27M or G34R) or a gain of function mutant isocitrate dehydrogenase IDH1R132H in combination with the loss of the histone remodeling factor ATRX and/or p53 tumor suppressor. We used a combination of these oncogenic hits to create a model of tumor engineering representing the first 3D human tissue model of GBM. This model combines the accurate multi-lineage differentiation and physiology of human brain with the easiness of manipulation of in vitro systems. H&E staining of the modified organoids shows remarkable dysplasia reminiscent of human primary glioma. Different genetic hits result in different type of dysplasia with IDH1R132H showing distinct morphology, suggestive of a broad range of applicability to model oncogenic-dependence in pediatric GBM. Cerebral organoids may also be used as a model to study tumor invasion starting with glioma stem-like cells (GSCs, which are believed to be the tumor-initiating cells in GBM). Indeed 923 GSCs (which are not invasive in mice brain) quickly and spontaneously (within few days) invade a co-cultured cerebral organoid. This study is the first to engineer a human CNS tumor in vitro. The model carries a tremendous potential to uncover the pathogenesis and initiation of brain cancer in a pediatric setting. Note: This abstract was not presented at the conference. Citation Format: Amin Ismail. A human mini brain model of pediatric GBM. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A15.

The hematopoietic regulator, ELF-1, enhances the transcriptional response to Interferon-β of the OAS1 anti-viral gene.

Interferon (IFN) therapy is effective in treating cancers, haematological and virus induced diseases. The classical Jak/Stat pathway of IFN signal transduction leading to changes in transcriptional activity is well established but alone does not explain the whole spectrum of cellular responses to IFN. Gene promoters contain cis-acting sequences that allow precise and contextual binding of transcription factors, which control gene expression. Using the transcriptional response to IFN as a starting point we report a high frequency of tandem GGAA motifs in the proximal promoters of Interferon stimulated genes, suggesting a key regulatory action. Utilizing the well-characterized anti-viral gene, OAS1, as an example Interferon stimulated gene promoter containing such a duplicated GGAA motif, we have demonstrated a regulatory role of this promoter in response to IFN by mutation analysis. Furthermore, we identified ELF-1 as a direct binding factor at this motif. Additionally, recruitment of RB1 and SP1 factors to the promoter following IFN stimulation is shown. ELF-1 overexpression enhanced and knockdown of ELF-1 inhibited full activation of OAS1 by IFN stimulation. Collectively, ELF-1 binds an important duplicated GGAA cis-acting element at the OAS1 promoter and in cooperation with RB1 and SP1 recruitment contributes to regulation in response to IFN stimulation.

SNP55, a new functional polymorphism of MDM2-P2 promoter, contributes to allele-specific expression of MDM2 in endometrial cancers.

BackgroundThe functional single nucleotide polymorphism (SNP) in the MDM2 promoter region, SNP309, is known to be associated with various diseases, particularly cancer. Although many studies have been performed to demonstrate the mechanism of allele-specific expression (ASE) on SNP309, they have only utilized in vitro techniques. It is unknown whether ASE of MDM2 is ascribed solely to SNP309, in vivo.MethodsWe attempted to evaluate ASE of MDM2 in vivo using post-labeling followed by automated capillary electrophoresis under single-strand conformation polymorphism conditions. For measuring a quantitative difference, we utilized the SNPs on the exons of MDM2 as markers, the status of which was heterozygous in a large population. To address the cause of ASE beyond 20 %, we confirmed sequences of both MDM2-3’UTR and promoter regions. We assessed the SNP which might be the cause of ASE using biomolecular interaction analysis and luciferase assay.ResultsASE beyond 20 % was detected in endometrial cancers, but not in cancer-free endometria samples only when an SNP rs1690916 was used as a marker. We suspected that this ASE in endometrial cancer was caused by the sequence heterogeneity in the MDM2-P2 promoter, and found a new functional polymorphism, which we labelled SNP55. There was no difference between cancer-free endometria and endometrial cancer samples neither for SNP55 genotype frequencies nor allele frequencies, and so, SNP55 alone does not affect endometrial cancer risk. The SNP55 status affected the DNA binding affinity of transcription factor Sp1 and nuclear factor kappa-B (NFκB). Transcriptional activity of the P2 promoter containing SNP55C was suppressed by NFκB p50 homodimers, but that of SNP55T was not. Only ASE-positive endometrial cancer samples displayed nuclear localization of NFκB p50.ConclusionsOur findings suggest that both the SNP55 status and the NFκB p50 activity are important in the transcriptional regulation of MDM2 in endometrial cancers.

Study on effect of lycorine in inducing apoptosis of pulmonary carcinoma cell A549

To explore the effect of lycorine in inducing apoptosis of pulmonary carcinoma cell A549 and its mechanism. In the study, pulmonary carcinoma cell A549 were taken as the experimental subject and processed with different concentrations of lycorine (0, 0.5, 1.0, 2.0, 4.0 and 8.0 μmol x L(-1)). The MTT method was used to observe the cell proliferation. The apoptosis rate of A549 cells was determined by Annexin FITC/PI double staining. The microplate reader was used to detect the activities of Bcl-2, Bax and p53. The changes in mitochondrial membrane potential were measured by the flow cytometry. The expressions of apoptosis-related factors Bcl-2, Bax, p53 and Survivin were determined by Real-time PCR. The results showed that lycorine significantly inhibited the proliferation of A549 cells (P < 0.05), induced the apoptosis on A549 cells (P < 0.05), increased the activities of Bax and p53, reduced Bcl-2 activity and mitochondrial membrane potential, and notably changed the gene expressions of Bcl-2, Bax, p53 and Survivin (P < 0.05). In conclusion, lycorine can induce the apoptosis of A549 cells and be applied to treat pulmonary carcinoma. Its mechanism may be related to the activation of relevant factors in Bcl-2 signaling pathway.

Abstract POSTER-TECH-1107: Primary platinum sensitivity in ovarian cancer and serum antibodies recognizing hypoxia-inducible factor 1-alpha and p53

Abstract Objective: Overexpression of hypoxia inducible factor 1-alpha (HIF-1α), a protein associated with apoptosis resistance, angiogenesis and metastasis, correlates with aberrant p53 protein accumulation in ovarian cancer. Humoral immunity to tumor-associated antigens in ovarian cancer has been associated with prognosis. We hypothesize that serum IgG antibodies recognizing HIF-1α and p53 in ovarian cancer patients could predict primary platinum response in ovarian cancer. Methods: Serum samples collected from ovarian cancer patients (n = 171) at the time of their initial surgery were analyzed by enzyme-linked immunosorbent assay for IgG antibodies to human recombinant HIF-1α and p53 proteins. The positive cut-off for each IgG antibody was derived at a serum level yielding highest hazard ratio of overall survival. Age, stage, grade, histology, neo-adjuvant therapy, surgical outcome, primary platinum response, and overall survival (OS) were correlated with serum IgG antibody levels. Results: Ovarian cancer patients were mostly stage III/IV (94%) with serous histology (85%), and the majority were optimally debulked (74%). Primary platinum sensitive patients (n = 116) had significantly higher IgG antibody levels than primary platinum resistant patients (n = 55) (median 0.83 µg/mL vs. 0.0 µg/mL, p = 0.001 for HIF-1α and median 6.37 µg/mL vs. 2.69 µg/mL, p =0.001 for p53). Humoral immune competence among cancer patients was not significantly different between primary platinum sensitive and resistant groups. Multivariate analysis of overall survival associated age and advanced stage with adverse survival, while elevated levels of serum HIF-1α IgG(≥ 0.32 µg/mL, HR = 0.57, p = 0.012) and p53 IgG (≥ 2.8 µg/mL, HR = 0.58, p = 0.024) were favorable prognostic factors. Multivariate logistic regression revealed HIF-1α and p53 -specific IgG antibody levels to be independent predictors of primary platinum sensitivity (OR=3.28, 95% CI 1.55 – 6.94, p = 0.002 for HIF-1α and OR = 4.61, 95% CI 2.11 – 10.1, p &amp;lt; 0.001 for p53). A model predicting sensitivity to adjuvant platinum-based treatment that includes only 2 factors, HIF-1α and p53 IgG antibody levels, yields an AUC = 0.710 and bests one containing 6 clinical variables (age, stage, histology, grade, surgical outcome, neoadjuvant treatment) which reaches AUC = 0.688. Our selected model of 4 factors: HIF-1α and p53 IgG antibody levels, stage, and neoadjuvant treatment, achieves comparable discrimination (AUC = 0.797) with a comprehensive model of 2 serum biomarkers and 6 clinical factors (AUC = 0.802). Conclusion: Viable strategies for the primary treatment of ovarian cancer have expanded beyond intravenous carboplatin and paclitaxel every 3 weeks after primary cytoreductive surgery to include alternate dosing, neoadjuvant chemotherapy, intraperitoneal administration, and the addition of novel biologic agents such as bevacizumab, yet there are no biomarkers to guide which patients derive the most benefit from these treatments, despite significant differences in their toxicities, cost, and associated quality of life. The ability of serum antibodies recognizing tumor associated antigens at time of diagnosis to identify patients most likely to achieve a durable response to platinum based chemotherapy may allow the selection of primary treatment to be optimized and improve response rates. Citation Format: M. D. Dao, K. J. Ovenell, K. J. Agnew, E. M. Swisher, B. A. Goff, M. L. Disis, J. B. Liao. Primary platinum sensitivity in ovarian cancer and serum antibodies recognizing hypoxia-inducible factor 1-alpha and p53 [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1107.

A suppressive effect of Sp1 recruitment to the first leader 5′ splice site region on L4-22K-mediated activation of the adenovirus major late promoter

Transcription from the adenovirus major late promoter (MLP) requires binding of late phase-specific factors to the so-called DE element located approximately 100 base pairs downstream of the MLP transcriptional start site. The adenovirus L4-22K protein binds to the DE element and stimulates transcription from the MLP via a DE sequence-dependent mechanism. Here we use a transient expression approach to show that L4-22K binds to an additional site downstream of the MLP start site, the so-called R1 region, which includes the major late first leader 5′ splice site. Binding of L4-22K to R1 has a suppressive effect on MLP transcription. L4-22K binds to the distal part of R1 and stimulates the recruitment of Sp1 and other cellular factors to a site overlapping the first leader 5′ splice site. Binding of Sp1 to the 5′ splice site region had an inhibitory effect on L4-22K-activated MLP transcription.

Abstract 3799: Cordyceptin induces apoptosis through repressing hTERT expression and inducing extranuclear export of hTERT

Abstract Cordycepin is an adenosine analog originally extracted from Cordyceps militaris that possesses many pharmacological effects including immune activation and antioxidant and antitumor effects. However, the underlying relationship between apoptosis and telomerase activity in response to cordycepin exposure has not been investigated. In this study, we found that cordycepin-induced apoptosis of human leukemia cells (H937 and THP-1 cells) was associated with inactivation of telomerase and downregulation of human telomerase reverse transcriptase (hTERT) as well as the transcription factors c-Myc and Sp1, which are required for basal transcription from the hTERT gene promoter. Cordycepin also attenuated the activation of phosphoinositide-3-kinase (PI3K)/Akt signaling, thereby reducing phosphorylation and nuclear translocation of hTERT. We further showed that the PI3K inhibitor LY29004 significantly decreased telomerase activity in cordycepin-treated cells and increased cordycepin-induced cell death. These findings demonstrate that cordycepin is cytotoxic to human leukemia cells and suppresses telomerase activity through transcriptional and post-translational suppression of hTERT by inactivating the PI3K/Akt signaling pathway. Citation Format: Nam Deuk Kim, Gi-Young Kim, Yung Hyun Choi. Cordyceptin induces apoptosis through repressing hTERT expression and inducing extranuclear export of hTERT. [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 3799. doi:10.1158/1538-7445.AM2015-3799

Abstract LB-138: Use of cerebral organoids to model pediatric gliomas with H3.3K27M and H3.3G34R

Abstract Glioblastoma (GBM) is the most devastating brain cancer with dismal prognosis both in adults and children and accounts for 16% of primary brain tumors. Despite aggressive therapeutic approaches, the majority of patients die within one year after diagnosis. Giving the limitation of current GBM mouse models, which do not reflect the biological properties of human tumors, in particular, tumor heterogeneity and pathogenesis, there is a growing need for new models. Current in vitro 3D models involve the use of hydrogel scaffolds and cell lines, which do not represent a viable developmental context where the tumor mass interacts with and invades the surrounding brain tissues. Starting with pluripotent human embryonic stem cells we reproduced a 3D tissue structure (so-called cerebral organoid) representing a developing human brain in vitro. We engineered these mini-brains to express oncogenic hits responsible for the derivation of tumors in pediatric GBM patients. Pediatric GBM is driven by a combination of epigenetic and genetic hits including the expression of a mutant histone H3 variant H3F3A (mutated at K27M or G34R) or a gain of function mutant isocitrate dehydrogenase IDH1R132H in combination with the loss of the histone remodeling factor ATRX and/or p53 tumor suppressor. We used a combination of these oncogenic hits to create a model of tumor engineering representing the first 3D human tissue model of GBM. This model combines the accurate multi-lineage differentiation and physiology of human brain with the easiness of manipulation of in vitro systems. H&amp;E staining of the modified organoids shows remarkable dysplasia reminiscent of human primary glioma. Different genetic hits result in different type of dysplasia with IDH1R132H showing distinct morphology, suggestive of a broad range of applicability to model oncogenic-dependence in pediatric GBM. Cerebral organoids may also be used as a model to study tumor invasion starting with glioma stem-like cells (GSCs, which are believed to be the tumor-initiating cells in GBM). Indeed 923 GSCs (which are not invasive in mice brain) quickly and spontaneously (within few days) invade a co-cultured cerebral organoid. This study is the first to engineer a human CNS tumor in vitro. The model carries a tremendous potential to uncover the pathogenesis and initiation of brain cancer in pediatric settings. Citation Format: Amin Ismail. Use of cerebral organoids to model pediatric gliomas with H3.3K27M and H3.3G34R. [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 LB-138. doi:10.1158/1538-7445.AM2015-LB-138

Abstract 4333: Association of TP53 mutation status with clinical outcomes in patients with EGFR mutated non-small cell lung cancer (NSCLC)

Abstract Introduction: Molecular characterization of tumors is critical in the modern treatment of NSCLC. Epidermal Growth Factor Receptor (EGFR) and p53 are commonly mutated genes in NSCLC with independent prognostic implications, but the significance of the co-existence of these mutations inpatients (pts) with Stage IV NSCLC is not well established. Herein, we describe the relationship of EGFR and TP53 mutations (MT) in pts with Stage 4 or recurrent NSCLC and their correlation with pt demographics and therapeutic outcomes. Study Design: We conducted a retrospective analysis of Stage 4 and recurrent EGFR MT (+) NSCLC pts with TP53 MT status determined by the Gene Sequence Analysis Panel obtained at the Abramson Cancer Center between 6/1/2012 and 2/1/2014. Overall survival (OS) and progression-free survival (PFS) were estimated for the TP53 MT (+) and wild type (WT) cohorts from the date of diagnosis. Results: Thirty-six EGFR (+) pts were identified, of whom 26 (72%) were TP53 MT (+). Pt characteristics were similar between cohorts, except for a higher rate of bone metastasis in pts with p53 WT (p = 0.07, Table 1). Median potential follow-up for OS was 15.1 mos. 60% of patients in TP53 WT group received TKI compared to 69% in TP53 MT (+). Of the 36 pts, 9 (25%). have died. A trend towards shorter median OS was seen in pts with p53 MT. (p = 0.08). PFS did not correlate with p53 MT status (p = 0.53). Conclusion: Coexistence of p53 MT and EGFR MT may be associated with shorter survival. Larger sample size and longer clinical follow up will be necessary to determine if these differences are statistically significant and whether TP53 MT status is an independent prognostic factor in EGFR MT NSCLC. Citation Format: Saman Ahmed, Charu Aggarwal, Rosemarie Mick, Joshua Bauml, Roger Cohen, Stephan Kadauke, Trisha Sterlicchi, Tracey L. Evans, John C. Kucharczuk, Charu Desphande, Jennifer J D Morrissette, Robert Daber, Corey J. Langer. Association of TP53 mutation status with clinical outcomes in patients with EGFR mutated non-small cell lung cancer (NSCLC). [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 4333. doi:10.1158/1538-7445.AM2015-4333

Abstract 1261: Comprehensive failure of intracellular protein homeostasis kills myeloma and solid cancer cells following VCP/p97 inhibition

Abstract Introduction: Intracellular protein homeostasis requires a well-controlled protein degradation machinery to clear damaged and old proteins and to replenish intracellular amino acid pools. Cancer cells may be particularly susceptible to agents that disrupt protein degradation because of unbalanced protein levels related to gain or loss of genetic material, high protein turnover, or high-level production of specific proteins such as Ig in multiple myeloma (MM). The AAA ATPase VCP (p97) is a master regulator of protein degradation that has been implicated in oncogenesis. Small molecule VCP inhibition (VCP-i) rapidly activates caspases in cancer cells, induces endoplasmic reticulum (ER) stress, and has anti-tumor activity in murine xenograft models. A phase 1 trial of VCP-i in relapsed MM has recently opened. Methods: We investigated the cellular mechanisms that govern VCP-i mediated cancer cell death in MM cell lines including bortezomib-adapted AMO1 cells, MM cells from patients with bortezomib-resistant MM, as well as lung cancer (A549) and osteosarcoma (Saos2) cells. Results: The ATP-competitive inhibitor DBeQ and the allosteric inhibitor NMS873 induced cell line as well as primary MM cell death at similar low micromolar concentrations independently of bortezomib sensitivity. DBeQ and NMS873 caused phosphorylation of eIF2alpha in a time- and dose-dependent manner and resulted in strong transcriptional and translational up-regulation of ATF4, CHOP and GADD34. VCP-i also increased expression of ER chaperones BiP and p58IPK. Inhibition of eIF2alpha de-phosphorylation with guanabenz reduced S6 phosphorylation, a marker of protein translation, and increased A549 and OPM2 cell survival early (8h) after VCP-i. Direct inhibition of protein translation with cycloheximide also decreased early VCP-i mediated cell death. Using MEFs deficient in eIF2alpha kinases we show that eIF2alpha phosphorylation following VCP-i depends on both the unfolded protein response mediator PERK and the nutrient sensor GCN2. We found that DBeQ induces GCN2 phosphorylation in parallel with loss of mTORC1 signalling, induction of the key autophagy factor p62, and accumulation of LC3-II. DBeQ also induced a rapid decrease in free intracellular L-amino acids. Depletion of selected amino acids in the cell culture medium increased cell death and mRNA levels of CHOP and GADD34 following VCP-i with DBeQ or NMS873, but not following induction of ER stress with tunicamycin. Conclusion: Collectively, these data show that both ATP-competitive and allosteric VCP-i effectively kills cancer cells independently of bortezomib sensitivity. Disrupting VCP function induces early cell death via inappropriate eIF2alpha-regulated protein translation downstream of GADD34. VCP-i also depletes the intracellular free amino acid pool, resulting in cell death despite compensatory catabolic processes. Citation Format: Katarzyna Parzych, Sandra Loaiza, Tamara M. Chinn, Philippa May, Florentina Porsch, Anastasios Karadimitris, Christoph Driessen, Heather P. Harding, David Ron, Holger W. Auner. Comprehensive failure of intracellular protein homeostasis kills myeloma and solid cancer cells following VCP/p97 inhibition. [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 1261. doi:10.1158/1538-7445.AM2015-1261

Data integration reveals key homeostatic mechanisms following low dose radiation exposure

The goal of this study was to define pathways regulated by low dose radiation to understand how biological systems respond to subtle perturbations in their environment and prioritize pathways for human health assessment. Using an in vitro 3-D human full thickness skin model, we have examined the temporal response of dermal and epidermal layers to 10cGy X-ray using transcriptomic, proteomic, phosphoproteomic and metabolomic platforms. Bioinformatics analysis of each dataset independently revealed potential signaling mechanisms affected by low dose radiation, and integrating data shed additional insight into the mechanisms regulating low dose responses in human tissue. We examined direct interactions among datasets (top down approach) and defined several hubs as significant regulators, including transcription factors (YY1, MYC and CREB1), kinases (CDK2, PLK1) and a protease (MMP2). These data indicate a shift in response across time — with an increase in DNA repair, tissue remodeling and repression of cell proliferation acutely (24–72h). Pathway-based integration (bottom up approach) identified common molecular and pathway responses to low dose radiation, including oxidative stress, nitric oxide signaling and transcriptional regulation through the SP1 factor that would not have been identified by the individual data sets. Significant regulation of key downstream metabolites of nitrative stress was measured within these pathways. Among the features identified in our study, the regulation of MMP2 and SP1 was experimentally validated. Our results demonstrate the advantage of data integration to broadly define the pathways and networks that represent the mechanisms by which complex biological systems respond to perturbation.

Mirna-181a expression Lead to Longer Animal Survival and Slower Tumor-Growth Rate in Diffuse Large B-Cell Lymphoma Xenograft Models

Introduction: Diffuse large B-cell lymphoma (DLBCL) is subdivided into the germinal center B-like (GCB) and activated B cell-like (ABC) subtypes by gene expression profiling, and these subtypes exhibit different clinical outcomes and signaling pathway deregulations. Compared to the GCB, the ABC-DLBCL subtype displays a more aggressive clinical course and shorter patient survival. Constitutive nuclear factor kappa-B (NF-kB) activity is often associated with the ABC-DLBCL subtype, however recent studies suggest that NF-kB signaling activation is also observed to a lower extent in the GCB-DLBCL subtype (Lina Odqvist et al. 2014). miRNAs have diagnostic and prognostic value in disease classification, and growing evidence implicates miRNAs in tumorigenesis, tumor maintenance, and dissemination through their ability to modulate the expression of critical genes and signaling networks. We previously demonstrated that miRNA-181a expression correlates with longer survival in patients treated with R-CHOP, independent of established clinical and molecular predictors. However, the molecular and cellular mechanisms underlying the association between miRNA-181a expression and improved prognosis in DLBCL patients are currently unknown. Herein we analyzed the role of miRNA-181a in DLBCL pathogenesis.Results:Quantitative RT-PCR analyses demonstrate higher endogenous miRNA-181a levels in centroblasts than in plasmablasts. Concordantly, endogenous miRNA-181a levels were significantly higher in GCB DLBCL cell lines and primary tumors compared with ABC DLBCL. These expression differences could not be attributed to distinct DNA methylation signatures in the miRNA-181a promoters (Chromosomes 1, 9) or regulatory elements as analyzed by Mass Array Sequenom Epityping. In search for putative miRNA-181a targets we identified 5 genes (CARD11, NFKB1A (IKBα), NFKB1 (p105/p50), RELA (p65), and REL (CREL)) within the NF-kB signaling pathway. Analyses of these targets show a decrease in the levels of these proteins and mRNAs in ABC and GCB DLBCL cell lines ectopically expressing miRNA-181a compared with scramble control plasmid. Luciferase reporter analyses encoding the respective wild type or mutated 3′UTR sequences demonstrate direct and specific targeting of these transcripts with the exception of RELA. Analysis of the net effect of miRNA-181a on NF-kB signaling using NF-kB luciferase reporter demonstrate significant decrease in NF-kB signaling. Concordantly, anti-miRNA-181a transfection led to increased NF-kB luciferase reporter activity. Moreover, western blot analyses of cytoplasmic and nuclear fractions showed a decrease in the levels of the transcription factors CREL and p50 in both cellular compartments, a decrease in the binding to DNA at NF-kB binding motifs, and a consequent decrease in NF-kB target gene transcription in the miRNA-181a expressing cells compared with scramble control. Together these studies point to miRNA-181a-mediated repression of NF-kB signaling in DLBCLs. Ectopic miRNA-181a expression led to a decrease in cell proliferation and an increase in cell death in both DLBCL subtypes, but this effect was more pronounced in the ABC DLBCL cell lines. The miRNA-181a-mediated increase in cell apoptosis could not be rescued by BCL2 co-transfection, an anti-apoptotic protein that was previously established as a direct miRNA-181a target. Analyses of miRNA-181a effects in NOD/SCID mice demonstrated that in vivo miRNA-181a induction in GCB and ABC human DLBCL xenografts led to decreased tumor growth and significantly longer animal survival. Notably, survival was prolonged in both GCB and ABC DLBCL bearing animals. [Display omitted] Conclusions: miRNA-181a directly suppress the NF-kB signaling pathway and lead to increased tumor cell death in both DLBCL subtypes suggesting that NF-kB deregulation is present in both tumor subtypes. However, the lower miRNA-181a expression level in the ABC DLBCL subtype may contribute to the higher NF-kB signaling activity that is observed in this subtype. Furthermore, our study provides a plausible explanation for the association between high miRNA-181a expression and longer survival of DLBCL patients. DisclosuresNo relevant conflicts of interest to declare.

Hypoxia-induced glucose-6-phosphate dehydrogenase overexpression and -activation in pulmonary artery smooth muscle cells: implication in pulmonary hypertension.

Severe pulmonary hypertension is a debilitating disease with an alarmingly low 5-yr life expectancy. Hypoxia, one of the causes of pulmonary hypertension, elicits constriction and remodeling of the pulmonary arteries. We now know that pulmonary arterial remodeling is a consequence of hyperplasia and hypertrophy of pulmonary artery smooth muscle (PASM), endothelial, myofibroblast, and stem cells. However, our knowledge about the mechanisms that cause these cells to proliferate and hypertrophy in response to hypoxic stimuli is still incomplete, and, hence, the treatment for severe pulmonary arterial hypertension is inadequate. Here we demonstrate that the activity and expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, are increased in hypoxic PASM cells and in lungs of chronic hypoxic rats. G6PD overexpression and -activation is stimulated by H2O2. Increased G6PD activity contributes to PASM cell proliferation by increasing Sp1 and hypoxia-inducible factor 1α (HIF-1α), which directs the cells to synthesize less contractile (myocardin and SM22α) and more proliferative (cyclin A and phospho-histone H3) proteins. G6PD inhibition with dehydroepiandrosterone increased myocardin expression in remodeled pulmonary arteries of moderate and severe pulmonary hypertensive rats. These observations suggest that altered glucose metabolism and G6PD overactivation play a key role in switching the PASM cells from the contractile to synthetic phenotype by increasing Sp1 and HIF-1α, which suppresses myocardin, a key cofactor that maintains smooth muscle cell in contractile state, and increasing hypoxia-induced PASM cell growth, and hence contribute to pulmonary arterial remodeling and pathogenesis of pulmonary hypertension.

Abstract 620: Evidence for combination tamoxifen and betulinic acid to treat hormone responsive- and unresponsive breast cancer by attenuation of Pygopus expression

Abstract The presence of the estrogen and progesterone hormone receptors (ER and PR) in advanced breast cancer tumour nuclei is predictive for response to hormone disruptive therapy. However, the accuracy of ER and PR testing can be variable and although patients receive significant benefit from hormone therapy, they ultimately become refractory to treatment. The chromatin remodeling protein: human Pygopus2 (hPygo2), is important for oncogenic growth and cell cycle progression and may serve as a diagnostic or targetable biomarker for breast cancer. Here we demonstrate that ER-alpha (Erα)−Sp1 transcription factor (Sp1) complexes associate with chromatin at adjacent motifs within the hPygo2 promoter in both hormone receptor positive and negative breast cancer cells. Erα agonists induced, while antagonists inhibited hPygo2 mRNA and protein expression. Erα and Sp1 occupancy at the hPygo2 promoter required intact promoter binding sites and functional DNA binding domains. Estrogen (E2) increased, while Fulvestrant decreased, Erα binding to the hPygo2 promoter with no effect on Sp1. Furthermore, Sp1-directed siRNA reduced Sp1 occupancy at the hPygo2 promoter and decreased hPygo2 mRNA and protein expression in both Erα-expressing (+) and Erα-nonexpressing (-) cells. Betulinic acid, an Sp1 inhibitor, caused cell cycle arrest, reduced Sp1 binding to the hPygo2 promoter and reduced hPygo2 expression in Erα- cell lines. The activation of hPygo2 expression either cooperatively or independently by Erα and Sp1 suggests their potential diagnostic and therapeutic benefit for both hormone responsive and unresponsive breast cancer. Citation Format: Youlian Tzenov, Philip Andrews, Cathy Popadiuk, Kenneth R. Kao. Evidence for combination tamoxifen and betulinic acid to treat hormone responsive- and unresponsive breast cancer by attenuation of Pygopus expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 620. doi:10.1158/1538-7445.AM2014-620

Abstract 502: Sp1 and HIFs mediate synergistic activation of ICAM1 gene under serum starved hypoxia and promotes growth of ovarian cancer

Abstract Cancer tissues are generally exposed to severe hypoxia conditions due to sparse and aberrant vasculature. Hypoxia-inducible factors HIF-1α and HIF-2α (HIF1 and HIF2, respectively) are major transcription factors required for adaptive responses to hypoxia. HIFs complexed with arylhydrocarbon receptor nuclear translocator bind target genes and act as enhancer of transcriptional regulation. This conventional mechanism regulates genes such as VEGF, GLUT1, and HO to improve mild to severe hypoxia conditions. It is likely that various oxygen concentrations and their duration exist within tumor tissue. It appears that adaptive response by HIFs is a major way to overcome stresses under milder hypoxia. However, majority of tumor tissues of many cancer types are known to undergo more severe hypoxic conditions. Thus, additional molecular mechanisms supporting HIFs signaling may be required to enhance survival activity of cancer cells. We have previously demonstrated that FVII gene, which is essential for initiation of extrinsic blood coagulation can be induced in ovarian clear cell carcinoma (CCC) cells in response to hypoxia. Activation of this gene potentially results in thrombotic events frequently observed in cancer patients. We have demonstrated that a physical interaction between Sp1 and HIF2 is a major cause of the activation of this gene under hypoxia. Furthermore, FVII activation is synergistically enhanced when cells are cultured under ischemic condition, in which hypoxia and serum-free conditions coexist. We envisioned that genes regulated by this mechanism are responsible for adaptive response to hypoxia with severity and/or duration characteristic to CCC tissues. Furthermore, ovarian cancer is a most malignant among gynecological diseases. CCC is highly tolerant to chemotherapy and the prognosis is poor. Thus, we first explored genes those are regulated by both Sp1 and HIFs in CCC cells by cDNA microarray analysis. We found that Sp1 and hypoxia inducible factors activate multiple genes under the ischemic condition in CCC cells. Among genes which meet this criteria, ICAM1 is one of a most dramatically induced gene. This transcriptional induction involves activation of NFκB associated with mTOR and autonomously produced TNFα. We further performed xenograft tumor experiments to test whether ICAM1 can be induced and contributes to CCC progression in vivo. It was found that ICAM1 is dramatically induced within xenograft tumor tissues. Tumor growth was evidently suppressed when ICAM1 inducton was abrogated by shRNA transfection. Thus, interplay among transcription factors associated with serum deficiency results in cancer progression. This study highlights importance of adaptive response to serum-starved condition as to how cancer cells overcome severe hypoxic stress. Citation Format: Shiro Koizume, Shin Ito, Yoshiyasu Nakamura, Mitsuyo Yoshihara, Yasuo Takano, Yohei Miyagi. Sp1 and HIFs mediate synergistic activation of ICAM1 gene under serum starved hypoxia and promotes growth of ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 502. doi:10.1158/1538-7445.AM2014-502

Abstract 2754: Induction of Sp1 factor by CYP1B1 inhibits TRAIL-mediated apoptotic pathway

Abstract Human cytochrome P450 1B1 (CYP1B1) belongs to the CYP1 family and is known as a major enzyme for estradiol 4-hydroxylation. It has been reported that CYP1B1 expression is higher in the tumor tissues than the normal ones, especially in hormone-related cancers such as breast, ovarian and prostate. Previously, we identified that CYP1B1 induces cell proliferation by activation of Wnt/β-catenin signal pathway. To explore the role of CYP1B1 on cancer cell proliferation further, we investigated whether CYP1B1 blocks apoptotic pathways. Using Western blot and RT-PCR, the expression of TRAIL was examined in MCF-7 cells, and we found that CYP1B1 overexpression or treatment with a CYP1B1 inducer, 7, 12-dimethylbenz[α]anthracene (DMBA) caused suppression of TRAIL and its receptors (DR4 and DR5) expression. However, when cells were treated with CYP1B1 siRNA or a selective CYP1B1 inhibitor, tetramethoxystilbene (TMS), expression of TRAIL and its receptors were significantly increased. Sp1, a zinc finger-containing transcription factor, has been reported to suppress TRAIL gene expression by binding TRAIL promoter. Interestingly, CYP1B1 overexpression and DMBA induced Sp1 expression and TMS was able to prevent DMBA-induced Sp1 expression. Sp1 knockdown with siRNA suppressed c-FLIP, an inhibitor protein of TRAIL-mediated apoptosis while it enhanced TRAIL and DR4 expression. These results were reversed by Sp1 overexpession. Moreover, attenuation of Sp1 DNA binding activity using mithramycin A, a Sp1 binding inhibitor, resulted in recovery of TRAIL expression suppressed by DMBA. Taken together, these data suggest that CYP1B1 prevents apoptosis via TRAIL suppression and this is mediated by Sp1 induction. Citation Format: Yeo-Jung Kwon, Nahee Park, Sangyun Shin, Dong-Jin Ye, Mihye Hong, Young-Jin Chun. Induction of Sp1 factor by CYP1B1 inhibits TRAIL-mediated apoptotic pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2754. doi:10.1158/1538-7445.AM2014-2754

Abstract 572: Transcriptional Regulation Of Renal Dopamine D1 Receptor Function During Oxidative Stress

There exists a strong link between Oxidative stress, renal dopaminergic system and hypertension. It is reported that reactive oxygen species attenuate renal proximal tubular dopamine receptor (D1R) function which disrupts sodium regulation and leads to hypertension. The mechanisms for renal D1R dysfunction, however are not clear. Here in, we investigated the role of redox sensitive transcription factors AP1 and SP3 in transcriptional suppression of D1R gene function and subsequent D1R signaling. Human kidney proximal tubular ( HK2 ) cell were treated with a pro-oxidant L-buthionine sulfoximine ( BSO , 50 mM for 72 hr) with and without a polyphenol resveratrol (250 μM). Treatment of HK2 cells with BSO caused oxidative stress (malondialdehyde [MDA], nmol/mg protein: control: 0.13 ± 0.01, BSO: 0.20 ± .02) and significantly reduced D1R mRNA and membrane receptor number (fmol/mg protein; control—140.1 ± 5.6, BSO—81.7 ± 4.4). Incubation of HK2 cells with SKF38393, a D1R agonist, caused a concentration dependent inhibition of Na/K-ATPase ( 86 Rb uptake, nmol/mg protein, control: basal—9.2 ± 0.2, SKF (1 μM)—6.3 ± 0.1). However, SKF38393 failed to inhibit Na/K-ATPase in BSO-treated cells (BSO: basal—9.5 ± 0.3, SKF—8.6 ± 0.2). Cells incubated with BSO exhibited increased AP1 (c-fos) and SP3 nuclear expression. Resveratrol per se had no effect on oxidative milieu (MDA: 0.11 ± 0.01) or D1R expression (143.3 ± 4.4), but it mitigated BSO-induced oxidative stress (MDA: 0.13 ± 0.02), AP1—SP3 up-regulation and D1R down-regulation (133.6 ± 6.2) and dysfunction (resveratrol + BSO [ 86 Rb uptake]: basal—9.7 ±0.3, SKF—6.7 ± 0.4). Incubation of cells with AP1 or SP3 specific siRNA reduced the expression of respective protein by ~85% and abolished BSO-induced D1R dysfunction. Down-regulation of SP3 had no effect on BSO-mediated AP1 activation, however, AP1 siRNA blocked BSO-dependent SP3 up-regulation. In cells transfected with Nrf2 siRNA, resveratrol failed to block BSO-induced AP1—SP3 activation and D1R dysfunction. These data shows that oxidative stress via AP1—SP3 activation suppresses D1R transcription which leads to D1R dysfunction. Resveratrol activates Nrf2, mitigates oxidative stress, blocks AP1—SP3 signaling and prevents D1R down-regulation and dysfunction.