Dose-dependent Reduction In Proliferation Research Articles

PLK1 inhibitors as a new targeted treatment for adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is an aggressive malignancy with limited treatment options. Polo-like kinase 1 (PLK1) is a promising drug target; PLK1 inhibitors (PLK1i) have been investigated in solid cancers and are more effective in TP53-mutated cases. We evaluated PLK1 expression in ACC samples and the efficacy of two PLK1i in ACC cell lines with different genetic backgrounds. PLK1 protein expression was investigated by immunohistochemistry in tissue samples and correlated with clinical data. The efficacy of rigosertib (RGS), targeting RAS/PI3K, CDKs and PLKs, and poloxin (Pol), specifically targeting the PLK1 polo-box domain, was tested in TP53-mutated NCI-H295R, MUC-1, and CU-ACC2 cells and in TP53 wild-type CU-ACC1. Effects on proliferation, apoptosis, and viability were determined. PLK1 immunostaining was stronger in TP53-mutated ACC samples vs wild-type (P = 0.0017). High PLK1 expression together with TP53 mutations correlated with shorter progression-free survival (P= 0.041). NCI-H295R showed a time- and dose-dependent reduction in proliferation with both PLK1i (P< 0.05at 100 nM RGS and 30 µM Pol). In MUC-1, a less pronounced decrease was observed (P< 0.05at 1000 nM RGS and 100 µM Pol). 100 nM RGS increased apoptosis in NCI-H295R (P< 0.001), with no effect on MUC-1. CU-ACC2 apoptosis was induced only at high concentrations (P < 0.05 at 3000 nM RGS and 100 µM Pol), while proliferation decreased at 1000 nM RGS and 30 µM Pol. CU-ACC1 proliferation reduced, and apoptosis increased, only at 100 µM Pol. TP53-mutated ACC cell lines demonstrated better response to PLK1i than wild-type CU-ACC1. These data suggest PLK1i may be a promising targeted treatment of a subset of ACC patients, pre-selected according to tumour genetic signature.

Impairment of 7F2 osteoblast function by simulated partial gravity in a Random Positioning Machine

The multifaceted adverse effects of reduced gravity pose a significant challenge to human spaceflight. Previous studies have shown that bone formation by osteoblasts decreases under microgravity conditions, both real and simulated. However, the effects of partial gravity on osteoblasts’ function are less well understood. Utilizing the software-driven newer version of the Random Positioning Machine (RPMSW), we simulated levels of partial gravity relevant to future manned space missions: Mars (0.38 G), Moon (0.16 G), and microgravity (Micro, ~10−3 G). Short-term (6 days) culture yielded a dose-dependent reduction in proliferation and the enzymatic activity of alkaline phosphatase (ALP), while long-term studies (21 days) showed a distinct dose-dependent inhibition of mineralization. By contrast, expression levels of key osteogenic genes (Alkaline phosphatase, Runt-related Transcription Factor 2, Sparc/osteonectin) exhibited a threshold behavior: gene expression was significantly inhibited when the cells were exposed to Mars-simulating partial gravity, and this was not reduced further when the cells were cultured under simulated Moon or microgravity conditions. Our data suggest that impairment of cell function with decreasing simulated gravity levels is graded and that the threshold profile observed for reduced gene expression is distinct from the dose dependence observed for cell proliferation, ALP activity, and mineral deposition. Our study is of relevance, given the dearth of research into the effects of Lunar and Martian gravity for forthcoming space exploration.

Abstract P5-08-08: C/EBPβ antagonist peptide, ST101, as a novel therapeutic for breast cancer

Abstract CCAAT/Enhancer Binding Protein Beta (C/EBPβ) is an oncogenic transcription factor that is active in many cancers, where it drives the expression of factors that promote tumor survival and proliferation. In breast cancer, increased C/EBPβ expression is associated with a metastatic phenotype, high tumor grade and an overall poor prognosis (Zahnow, 2009). C/EBPβ forms dimers via leucine zipper domain interactions that are required for its transcriptional activity . ST101 is a novel peptide antagonist of C/EBPβ currently being evaluated in a Phase 1/2 clinical study in patients with advanced unresectable and metastatic solid tumors. ST101 binds to the C/EBPβ leucine zipper domain and antagonizes it’s dimerization, resulting in increased C/EBPβ proteosomal degredation and significantly decreased target gene transactivation. Here, we describe the non-clinical anti-tumor activity of ST101 against breast cancer. In vitro studies in 4T1-luc triple-negative (TNBC) and MCF7 HRPOS BC cells demonstrate ST101 dose-dependent cytotoxicity (EC50 of 4.8 and 2.1 µM, respectively), accompanied by significant impact on C/EBPβ-mediated gene expression as determined by qPCR analysis. In contrast, normal human mammary epithelial cells were not sensitive to ST101 (EC50 &amp;gt;100 µM). In HR-negative BC and TNBC tumoroids in vitro, ST101 exposure resulted in a dose-dependent reduction in proliferation and viability, with EC50 values of 18.56 and 15.32 µM, respectively. In vivo experiments indicate significant anti-tumor activity. In a MCF7 subcutaneous xenograft model, 25 mg/kg ST101 resulted in tumor growth delay and a significant decrease in tumor volume (p&amp;lt;0.001 vs. controls). In a 4T1-luc orthotopic model, ST101 resulted in dose-dependent tumor growth inhibition, and tumor regression following 25 mg/kg ST101 (p&amp;lt;0.001 vs. controls). These data emphasize the potential of ST101 as a potent peptide therapeutic for HRPOS and triple-negative BC. Citation Format: Jim Rotolo, Mark Koester, Rick Ramirez, Siok Leong, Lila Ghamsari, Erin Gallagher, Emad Darvishi, Gene Merutka, Barry Kappel. C/EBPβ antagonist peptide, ST101, as a novel therapeutic for breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-08.

The collagenase of the bacterium Clostridium histolyticum does not favor metastasis of breast cancer.

Breast cancer is the most common malignancy among women worldwide. As survival rates increase, breast reconstruction and quality of life gain importance. Of all women undergoing breast reconstruction, approximately, 70% opt for silicone implants and 50% of those develop capsular contracture, the most prevalent long-term complication. The collagenase of the bacterium Clostridium histolyticum (CCH) showed promising results in the therapy of capsule contracture; however, its influence on residual cancer cells is unknown. The aim of this study was to investigate whether CCH-treatment negatively impacts breast cancer cells in vitro and in vivo. MDA-MB-231 and MCF-7 cells were used in this study. In vitro, we tested the influence of CCH on proliferation, wound healing, migration and cell cycle by MTT-assay, scratch-assay, transwell-migration-assay, and flow cytometry. In vivo, solid tumors were induced in immune-deficient mice. CCH was injected into the tumors and tumor growth and metastasis formation was monitored by caliper measurement, in vivo bioluminescence imaging and histology. Gene expression analysis was performed by microarray including 27,190 genes. CCH-incubation led to a dose-dependent reduction in proliferation for both cell lines, while wound healing was reduced only in MDA-MB-231 cells. No morphological alterations were monitored in cell cycle or apoptosis. In vivo, bioluminescence imaging and histology did not show any evidence of metastasis. Although CCH led to changes in gene expression of breast cancer cells, no relevant alterations in metastasis-related genes were monitored. CCH has no impact on tumor growth or metastasis formation in vitro and in vivo. This paves the way for first clinical trials.

Screening of Health-Associated Oral Bacteria for Anticancer Properties in vitro

While extensive literature exists about the role of oral bacterial pathogens like Porphyromonas gingivalis and Fusobacterium nucleatum in oral squamous cell carcinoma (OSCC), the role of health-associated species has been largely unexplored. In this study, we assessed the effect of Streptococcus mitis, Rothia mucilaginosa, Neisseria flavescens, Haemophilus parainfluenzae, Lautropia mirabilis, and Veillonella parvula on proliferation and expression of marker genes (IL-6, TNF-α, MMP3, CD36, CCD1, and NANOG) in OSCC cell lines CAL27, SCC25, and SCC4. Porphyromonas gingivalis was included as a pathogenic control. Both bacterial lysates (3 concentrations) and live cells (3 MOIs) were tested. S. mitis, H. parainfluenzae, and N. flavescens resulted in substantial, dose-dependent reduction of proliferation, which was found to be mediated by H2O2 for the former and intracellular infection in the latter two species. However, only H. parainfluenzae showed differential antiproliferative effect against the cancer cell lines vs. the normal control (TIGKs). In the gene expression assays, the health-associated species mostly downregulated CD36, a gene that plays an important role in tumor growth and metastasis, while P. gingivalis upregulated it. IL6 and TNF expression, on the other hand, was upregulated by almost all species, particularly the Gram-negatives including P. gingivalis. The effect on other genes was less evident and varied significantly by cell line. This exploratory study is the first insight into how health-associated bacteria may interact with OSCC. Further studies to explore whether the observed effects may have implications for the prevention or treatment of oral cancer are warranted.

Abstract 3449: A novel rapid zebrafish model for validation of potential therapies for fatal pediatric brain tumors

Abstract Diffuse intrinsic pontine glioma (DIPG) and atypical teratoid/rhabdoid tumors (AT/RT) are deadly pediatric brain tumors. There is an urgent need of better therapeutics for improving survival and quality of life for these patients. Additionally, we need improved animal models to accelerate identification of novel therapeutics and improve through-put. In line with our long-term interests of validating therapies and developing model systems for pediatric brain tumors, we tested a repurposed drug quinacrine as a potential therapy using a new and faster model system in zebrafish. Quinacrine is a safe and widely used treatment for pediatric malaria and parasitic infections. We hypothesized that quinacrine will increase tumor cell death and decrease tumorigenicity of DIPG and ATRT. We used quinacrine in six patient-derived cell lines: three AT/RT (BT37, CHLA-05, CHLA-266) and three DIPG (JHHDIPG1, SUDIPGXIII, SF7761). In all tumor cell lines, quinacrine caused a dose-dependent reduction in proliferation (BrdU) and increase in apoptosis (cleaved caspase-3 and cleaved PARP) compared to vehicle-treated cells (P&amp;lt;0.01). Quinacrine had no effect on growth of normal hindbrain neural stem and progenitor cells. Using quinacrine fluorescence as a surrogate, we achieved micromolar concentration of quinacrine in the mouse and zebrafish brain without overt toxicity. Treatment of mice bearing ATRT flank tumors with quinacrine resulted in decreased tumor volume compared to vehicle-treated mice (P&amp;lt;0.05). To validate quinacrine orthotopically in DIPG, we developed a novel model in zebrafish wherein we injected fluorescent DIPG cells into the developing zebrafish blastula. Two days post injection, DIPG cells had homed to the zebrafish brain. Treatment of xenografted zebrafish with quinacrine for 72 hours decreased DIPG growth by 40% as measured by fluorescence, suggesting that minor groove binding drugs like quinacrine are a viable treatment strategy for these tumors. Using this new system, we were able to validate a potential therapeutic in 7 days versus 7 months in mice. Future studies are aimed at investigating the mechanism of quinacrine in these tumors and optimizing our zebrafish model for high-throughput screening of potential drugs. Citation Format: Harpreet Kaur, Huizi Guo, Kevin Emmerich, David White, Peter Green, Alara Michelle Hector, Sepehr Akhtarkhavari, Anukriti Bhargava, Allison Martin, Smit Shah, Charles G. Eberhart, Jeffrey Mumm, Eric H. Raabe. A novel rapid zebrafish model for validation of potential therapies for fatal pediatric brain tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3449.

Abstract A38: Validation of potential therapies for treatment of fatal pediatric brain tumors DIPG and AT/RT using a novel rapid intracranial model in zebrafish

Abstract Diffuse intrinsic pontine glioma (DIPG) and atypical teratoid/rhabdoid tumors (AT/RT) are deadly pediatric brain tumors. Developing new targets and therapeutics are urgently needed. We have previously shown that multiple primary brain tumors and cell lines express increased amounts of the epigenetic modifier and DNA binding oncoprotein high-mobility group AT-hook 2 (HMGA2). Targeting HMGA2 using short hairpins decreased AT/RT and glioma growth in xenografted mice. We hypothesized that pharmacologic inhibition of HMGA proteins using DNA minor-groove binding drugs such as quinacrine would decrease tumor growth due to displacement of HMGA proteins from DNA. Quinacrine is a safe and widely used treatment for pediatric malaria and parasitic infections. We used quinacrine in six patient-derived cell lines: three AT/RT (BT37, CHLA-05, CHLA-266) and three DIPG (JHHDIPG1, SUDIPGXIII, SF7761). Using quinacrine fluorescence as a surrogate, we can achieve micromolar concentration of quinacrine in the mouse and zebrafish brain after oral administration without overt toxicity. In both tumor cell lines, quinacrine causes a dose-dependent reduction in proliferation (BrdU) and increase in apoptosis (cleaved caspase-3 and cleaved PARP) compared to vehicle-treated cells (P&amp;lt;0.01). Quinacrine had no effect on growth of normal hindbrain neural stem and progenitor cells. Treatment of mice bearing ATRT flank tumors with quinacrine resulted in decreased tumor volume compared to vehicle-treated mice (P&amp;lt;0.05). Current mouse intracranial DIPG models can take 6-9 months due to long latency. To validate quinacrine orthotopically in DIPG, we developed a novel model in zebrafish wherein we injected fluorescent DIPG cells into the developing zebrafish blastula. Five days post injection, DIPG cells had homed to the zebrafish brain. Treatment of xenografted zebrafish with quinacrine for 48 hours decreased DIPG growth by 40% as measured by fluorescence, suggesting that minor groove binding drugs such as quinacrine are a viable treatment strategy for these tumors. Future studies are aimed at investigating the mechanism of quinacrine in these tumors. Citation Format: Harpreet Kaur, Huizi Guo, David White, Peter Green, Alara M. Hector, Sepehr Akhtarkhavari, Anukriti Bhargava, Smit Shah, Charles G. Eberhart, Jeffrey Mumm, Eric H. Raabe. Validation of potential therapies for treatment of fatal pediatric brain tumors DIPG and AT/RT using a novel rapid intracranial model in zebrafish [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A38.

Caspase mediated cytotoxicity of a yellow pigment produced by Exiguobacterium alkaliphilum on human cancer cell lines

Context: Investigation of microbial diversity for the identification of novel bioactive compounds or therapeutics is a more potential and incessant ongoing process for drug development research. Many tumors are generally metastasized at the time of diagnosis and restorative surgery is rarely achieved. As response rates to chemotherapy are low, surgery remains the only effective treatment. Consequently, a substantial need for new therapeutic options is essential. The promotion of apoptosis in cancer cells could potentially lead to the regression and improved prognosis of cancer. Aims: To assess the in vitro anticancer activity of the pigment extracted from E. alkaliphilum on cancer cell lines. Methods: The effect of a pigmented compound produced by Exiguobacterium alkaliphilum, isolated from a soil sample was investigated on various human cancer cell lines. Proliferation and viability were analyzed using cell counting and MTT cell proliferation assay. Quantification of lactate dehydrogenase (LDH), a cytosolic enzyme, which is an indicator of cellular toxicity was carried out. Apoptosis was determined by DNA fragmentation, and by colorimetric assays for caspases 3, 7 and 10. The cell cycle analysis was also performed using a flow cytometer, by fixing the cells with propidium iodide. The structural elucidation of the active compound was analyzed by GC-MS. Results: Pigment extracted from E. alkaliphilum showed a time and dose-dependent reduction of proliferation in HepG2, HeLa, MCF7, Jurkat and K562 cell lines with lower IC50 concentrations and induction of apoptosis was significantly evident from the caspase assay and DNA fragmentation. Conclusions: The pigment showed a significant anticancer property on cancer cell lines and this study thereby provides a rationale for pre-clinical and clinical evaluation for the effective treatment of cancer.

Screening of five Sri Lankan endemic plants for anti-cancer effects on breast cancer stem cells isolated from MCF-7 and MDA-MB-231 cell lines

Purpose: To screen selected endemic plants grown in Sri Lanka on breast cancer stem cells (bCSCs) for their anti-cancer propertiesMethods: Breast-CSCs expressing CD24-/CD44+ surface markers were isolated from MDA-MB-231 cells by magnetic-assisted cell sorting method and validated using flow cytometry. A panel of forty extracts from barks and leaves of Doona nervosa, Garcinia quaesita, Garcinia zeylanica, Memecylon rostratum and Schumacheria castaneifolia were obtained by sequential solvent extraction and tested on bCSC-mammospheres derived from MDA-MB-231 and MCF-7 cells and normal mammary epithelial (MCF-10A) cells. Proliferation and cell stemness were analyzed using WST-1 cell proliferation assay and alkaline phosphatase assay.Results: Hexane and chloroform extracts of G. zeylanica and G. quaesita barks showed dosedependent reduction in proliferation and stemness in both bCSCs tested with less effect on MCF-10A cells. Hexane, chloroform and ethyl acetate extracts of S. castaneifolia bark selectively inhibited mammospheres of triple negative bCSCs cells.Conclusion: This study demonstrates that the non-polar extracts of G. zeylanica and G. quaesita, S. castaneifolia barks inhibit the proliferation of bCSCs of triple negative and estrogen-progesterone positive breast cancers. Findings of the present study may useful for developing a future anti-cancer therapeutics which can target bCSCs.Keywords: Cancer stem cells, Garcinia zeylanica, Garcinia quaesita, Schumacheria castaneifolia, Mammospheres

Abstract 3815: Cyclin-dependent kinase 9: a potential therapeutic target for esophageal adenocarcinoma

Abstract Objective: Major limitation of successful implementation of targeted therapy in esophageal adenocarcinoma (EAC) is low frequency of target specific biomarker alteration and intratumoral heterogeneity. Cyclin dependent kinase 9 (CDK9) is a ubiquitous serine threonine kinase whoseinhibition has been used as a therapeutic option in other solid tumors. However, critical in vitro and in vivo information about the efficacy and mechanism of CDK9 inhibition in EAC is lacking and needs to be generated before a clinical trial is proposed. Design: We assessed the CDK9 protein expression in EAC cell lines and in patient tissue samples of Barrett's esophagus and EAC. Subsequently, we studied effects of genetic downregulation (shCDK9) in SKGT4 and chemical inhibition of CDK9 by CDK9 inhibitor II, a specific, potent competitive inhibitor of CDK9 (Santa Cruz Biotechnology, CA) on proliferation, apoptosis, and cell cycle of SKGT4, FLO-1 and OE33. In vivo effects of shCDK9 and CDK9 inhibitor II was analyzed in xenografts experiments performed on nude mice after injection of FLO-1 or shCDK9-SKGT 4 and control cells in right flank or abdomen. We also assessed the effects of shCDK9 and CDK9 inhibitor II on expression of MCL-1 and phosphorylation of RNA polymerase II in EAC cells. Results: Higher expression of CDK9 was observed in all EAC cell lines and in tumor samples of patients with EAC as compared to normal squamous epithelium and Barrett's Esophagus. shCDK9 in SKGT4 cells induced significant reduction in proliferation, increase in apoptosis and G1 arrest as compared to control cells in vitro. In vivo experiment with SKGT4-shCDK9 demonstrated no tumor growth in 4 of 10 mice and tumor volume was significantly smaller in SKGT4-shCDK9 as compared to control SKGT4 cells (72.89 ± 12.88 mm3 v.270 ± 64.07 mm3, p&amp;lt; 0.01) in other mice. Treatment with CDK9 inhibitor II demonstrated dose dependent reduction in proliferation, increased apoptosis and G1 arrest in all three cell lines in vitro and more than 50% reduction in tumor volume in xenografts as compared to control. CDK9 inhibitor II reduced RNA Pol II phosphorylation and expression of MCL-l and up-regulation of MCL-1 expression induced resistance to CDK9 inhibitor II in all three EAC cell lines.. Conclusions: CDK9 inhibition has strong anti-EAC properties in vitro and in vivo. These properties along with strong diffuse expression of CDK9 in human EAC support further exploration of CDK9 as a potential therapeutic target for EAC. Citation Format: Zhimin Tong, Devkumar Chatterjee, Defeng Deng, Omkara Veeranki, Alicia Mejia, Jaime Rodriguez, Jaffer Ajani, Wayne Hofstetter, Steven Lin, Sushovan Guha, Scott Kopetz, Sunil Krishnan, Dipen Maru. Cyclin-dependent kinase 9: a potential therapeutic target for esophageal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3815.

Mediator Kinase Inhibition Triggers a Phenotypic Switch from Neonatal to Adult-like Megakaryopoiesis

Several recent studies have demonstrated substantial biological differences between cord blood (CB)- and adult peripheral blood (PB)-derived megakaryocytes (MKs). Specifically, neonatal (CB) progenitors proliferate at a much higher rate than adult (PB) progenitors, and generate 10-fold more MKs per progenitor when cultured with thrombopoietin (Tpo). The highly proliferative neonatal MKs undergo cytoplasmic maturation without polyploidization, which ultimately generates large numbers of small, low ploidy, but fully mature MKs. Adult MKs, in contrast, undergo successive rounds of endomitosis to reach much higher ploidy levels, and maturation is coupled with polyploidization, so that MKs with the highest ploidy levels are also the most mature. The molecular mechanisms underlying these developmental differences are just beginning to be elucidated. Here, we investigated the effects of cortistatin A (CA), a highly specific small molecule inhibitor of cyclin-dependent kinase 8 (CDK8) and its paralog CDK19, on megakaryopoiesis. CDK8 and CDK19, together with CCNC (Cyclin C), MED12/MED12L and MED13/MED13L, form "CDK modules" which can associate with Mediator, a 26-subunit complex that acts as a bridge between transcription factors and the transcriptional machinery to coordinate gene expression. The Mediator complex has been implicated in developmental disorders and cancer. CB-derived MK progenitors treated with CA from day 7 to day 14 of culture exhibited a dose-dependent reduction in proliferation (6.2±1.7 vs. 24.9±2.2 fold expansion in treated vs. control cultures; p=0.003), accompanied by an increase in ploidy levels to those comparable to adult PB-derived MKs (34±6% vs. 8.9±0.9% MKs with ploidy ≥8N in treated vs. control cultures; p=0.014). MK maturation, evaluated by CD42b surface expression level, also increased with advancing ploidy in CA-treated MKs, in a manner similar to that observed in adult MKs, and CA-treated mature MKs were capable of pro-platelet formation in vitro. These changes were not observed when undifferentiated CB-derived CD34+ or CD41-negative cells were treated, indicating that the effect was specific to committed MK progenitors. CA treatment induced the expected decrease in STAT-1 phosphorylation at Serine 727, a specific site of CDK8-mediated phosphorylation, confirming effective CDK8 inhibition in treated MKs. Next, we used microarray to evaluate the gene expression profile of CB-derived MKs following CA treatment for 4 and 8 hours vs. untreated cells. These studies revealed significant CA-induced changes in the MK gene expression profile. By Gene Set Enrichment Analysis (GSEA), CA treatment significantly upregulated genes that were downregulated in CB- vs. PB-derived MKs. Furthermore, genes upregulated by CA in CB-derived MKs were also upregulated in the megakaryoblastic cell line SET2 (Pelish, Liau et al., Nature, in press), suggesting that the gene expression program affected in SET2 cells is likely the same one affected in neonatal MKs. At the protein level, we observed time- and dose-dependent increases in RUNX-1 in CA-treated vs. control MKs. In summary, this study demonstrated that treatment with CA induced a phenotypic switch from neonatal to adult-like megakaryopoiesis, accompanied by changes in the MK gene expression profile. These findings indicate a novel role for Mediator kinases in the regulation of megakaryopoiesis, and potentially on the developmental differences between neonatal and adult MKs. These studies open the door to a better understanding of and to potential novel therapies for a number of developmental stage-specific megakaryocyte and platelet disorders, which exclusively or more severely affect neonates and infants, including the thrombocytopenia/absent radius (TAR) syndrome and the transient myeloproliferative disorder associated with trisomy 21 and GATA1s mutations. DisclosuresNo relevant conflicts of interest to declare.

Glucose transporter isoform 1 expression enhances metastasis of malignant melanoma cells.

The glucose transporter isoform 1 (GLUT1; SLC2A1) is a key rate-limiting factor in the transport of glucose into cancer cells. Enhanced GLUT1 expression and accelerated glycolysis have been found to promote aggressive growth in a range of tumor entities. However, it was unknown whether GLUT1 directly impacts metastasis. Here, we aimed at analyzing the expression and function of GLUT1 in malignant melanoma. Immunohistochemical analysis of 78 primary human melanomas on a tissue micro array showed that GLUT1 expression significantly correlated with the mitotic activity and a poor survival. To determine the functional role of GLUT1 in melanoma, we stably suppressed GLUT1 in the murine melanoma cell line B16 with shRNA. GLUT1 suppressed melanoma cells revealed significantly reduced proliferation, apoptosis resistance, migratory activity and matrix metalloproteinase 2 (MMP2) expression. In a syngeneic murine model of hepatic metastasis, GLUT1-suppressed cells formed significantly less metastases and showed increased apoptosis compared to metastases formed by control cells. Treatment of four different human melanoma cell lines with a pharmacological GLUT1 inhibitor caused a dose-dependent reduction of proliferation, apoptosis resistance, migratory activity and MMP2 expression. Analysis of MAPK signal pathways showed that GLUT1 inhibition significantly decreased JNK activation, which regulates a wide range of targets in the metastatic cascade. In summary, our study provides functional evidence that enhanced GLUT1 expression in melanoma cells favors their metastatic behavior. These findings specify GLUT1 as an attractive therapeutic target and prognostic marker for this highly aggressive tumor.

Electrophysiologic and molecular characteristics of cardiomyocytes after heavy ion irradiation in the frame of the ESA IBER-10 program

Since essentially no information is available on the effects of high linear energy transfer (LET) radiation on the heart, an assessment of possible late effects on the cardiovascular system is important with respect to the planning of manned long-term space missions [ 1, 2]. In order to examine if and to what extent heart muscle cells are affected by an exposure to heavy ions, primary avian cardiomyocytes were isolated, cultured in vitro and exposed at GSI (Darmstadt) to different ion species (carbon, titanium and nickel). Investigation of electrophysiologic radiation effects was performed on cells grown on a microelectrode array (MEA) allowing the monitoring of beat rate, spike shape, field action potential duration and signal conduction pathways across the electrode array. Data were analysed using the DrCell software [ 3]. In parallel, immunohistological stainings were conducted to examine cell cycle progression, DNA-damage repair and apoptosis.Our preliminary data indicate that primary cardiac cells possess a high robustness toward ionizing radiation as cultures receiving doses of up to 7 Gy still show unaltered electrophysiologic activity. This is exemplarily shown in Fig. 1, where the beat rate of cells irradiated with 2 and 7 Gy carbon ions has been plotted. Figure 1 also illustrates that within each treatment group, inter-sample variations are quite large and thus do not allow detection of subtle changes in cellular electrophysiology, probably due to the embryonic character of the cells.Interestingly, in terms of the formation of double-strand breaks (DSBs) and cell cycle progression delay, the cultures showed a dose-dependent reduction in proliferation and DSB accumulation.Taken together, our electrophysiologic studies indicate that the large variations within the same treatment group of avian embryonic cardiomyocytes obscure the detection of radiation effects. To account for this problem, we will use in further studies human cardiomyocytes differentiated from iPS cells, as these cells resemble more closely the condition of an adult human heart and do not undergo developmental changes as embryonic cell cultures do. Fig. 1.Normalized beat rates measured in samples before and after exposure to carbon ions. (spread-out Bragg peak, LET: 75 keV/µm). Average beat rates range from 22 to 53 beats/min.

Abstract 3249: Targeting the miR29b/Sp1/Dnmt pathway for curative therapy in Mll-PTD/Flt3-ITD acute myeloid leukemia.

Abstract The partial tandem duplication (PTD) of the MLL gene is a molecular defect in human myelodysplastic syndrome and acute myeloid leukemia (AML) and associates with poor patient outcomes, especially when other molecular prognostic markers are co-present such as the internal tandem duplication (ITD) of FLT3, i.e., FLT3-ITD. In human AML, MLL-PTD alters the epigenome via aberrant H3K4 methylation activity but is also associated with increased DNA methylation through an unknown mechanism (Whitman, et al, Blood 2008). Using a double knock-in mouse model of AML with Mll-PTD and Flt3-ITD, we analyzed the DNA methylome using a methyl binding protein (MBD2) pull down followed by next generation sequencing. Leukemic mouse bone marrow (BM, n=8) exhibited a 1.7 fold increase in global DNA methylation compared to non-leukemic controls (n=9, P=0.017). Consistent with this finding, DNA methyltransferases (Dmnt) 1, 3a, and 3b were overexpressed (1.5 fold P=0.03, 2.3 fold P=0.01, and 5.3 fold P=0.03, respectively). We previously showed that the non-coding microRNA 29b (miR-29b) expression directly downmodulates DNMT3a and 3b, but indirectly inhibits DNMT1 via miR-29b-driven SP1 suppression. In the murine Mll-PTD/Flt3-ITD AML, the precursor to the mature miR-29b (pri-miR-29b-2) was downregulated in leukemic BM (0.7-fold, P&amp;lt;0.001), whereas Sp1 mRNA was upregulated compared to wildtype (1.6-fold P=0.01). Bortezomib is a proteasome inhibitor that also downregulates Dnmt's (Liu, et al, Blood, 2008) by increasing the expression of miR-29b (Liu, et al, Cancer Cell, 2010). In vitro, bortezomib induced a dose-dependent reduction in proliferation of Mll-PTD/Flt3-ITD leukemic blasts (MTS assay, EC50 23.83nM) and apoptosis. Prior to cell death in bortezomib-treated cells, expression of pri-miR-29b-2 increased by 1.7 fold over vehicle treated cells (P=0.01) while Dnmt3b mRNA (P=0.001) and protein levels decreased to half of the vehicle treated level. Targeting of this pathway in vivo using liposomal-bortezomib (1mg/kg IV twice per week for 2 weeks followed by 2mg/kg IV twice per week for 2 weeks) significantly increased survival of AML transplanted mice, compared with vehicle or free-bortezomib with 80% alive 90 days post-transplant (P&amp;lt;0.001). At day 90, treated mice had normal spleen weights (mean 86mg) and absence of leukemic blasts in BM, liver, or spleen compared to vehicle-treated mice that showed significant blast infiltration (mean spleen weight 396mg, P&amp;lt;0.001). Taken together, these data support the miR-29b/SP1/DNMT pathway of epigenetic modulation as active in Mll-PTD/Flt3-ITD AML, and indicate bortezomib as a valuable treatment option to be explored in patients with MLL-PTD/FLT3-ITD-associated AML. Citation Format: Kelsie M. Bernot, John S. Nemer, Ramasamy Santhanam, Shujun Liu, Gabriel G. Marcucci, Nicholas A. Zorko, Susan P. Whitman, Pearlly Yan, David Frankhouser, Ralf Bundschuh, Mengzi Zhang, Ronald F. Siebenaler, Elshafa H. Ahmed, Kathleen K. McConnell, Maura Munoz, Daniel L. Brook, Adrienne M. Dorrance, Katy E. Dickerson, Xiaoli Zhang, Jianying Zhang, David Jarjoura, Robert Lee, William Blum, Michael A. Caligiuri, Guido Marcucci. Targeting the miR29b/Sp1/Dnmt pathway for curative therapy in Mll-PTD/Flt3-ITD acute myeloid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3249. doi:10.1158/1538-7445.AM2013-3249

Abstract 2006: Sequencing of mismatch repair deficient tumors identifies a synthetic lethal interaction with other DNA repair pathways.

Abstract DNA replication errors that persist as mismatch mutations make up the molecular fingerprint of mismatch repair (MMR) deficient tumors and convey them with different prognostic and therapeutic outcome upon chemotherapy. Using whole-genome and -exome sequencing, we here characterize the somatic mutation patterns underlying MMR-deficient tumors. In particular, we show that mutation frequencies are &amp;gt;30-fold increased relative to MMR-proficient tumors: insertion-deletion (indel) mutations are largely confined to homopolymers, whereas substitutions mainly consist of single basepair transitions located near indels. Since exonic indels result in loss-of-function frameshift mutations, indels were less frequent in the exome due to negative selection. However, a small fraction of exonic indels was positively selected and occurred as hotspot mutations, preferentially affecting the ‘DNA double-strand break (DSB) repair by homologous recombination (HR)’ pathway (on average, each MMR-deficient tumor contained 2.6 indels in this pathway). We quantified DSB repair by immunofluorescent staining of RAD51 foci in 8 MMR-deficient and 4 MMR-proficient tumor cultures in the presence of the PARP inhibitor olaparib. We did not observe a difference in RAD51 foci formation between MMR-deficient and MMR-proficient tumor cultures in the absence of olaparib (10±2% of cells versus 13±2% showed RAD51 foci formation, P=0.74). Upon 10μM olaparib exposure, MMR-deficient tumor cultures accumulated less RAD51-positive foci (19±3% in MMR-deficient versus 37±4% in MMR-proficient cells; P=0.02), thereby confirming that these tumors were at least partly deficient in DNA DSB repair by HR. We also investigated the degree of unrepaired DNA damage by H2AX immunofluorescence and olaparib drastically increased the number of H2AX foci in all cells regardless of their MMR status. Furthermore, MMR-deficient cultures exhibited a dose-dependent reduction in proliferation under olaparib, whereas MMR-proficient cells did not respond (P&amp;lt;0.001 by repeated measurement). On average, 1, 3 and 10μM of olaparib decreased proliferation of MMR-deficient cells by respectively 15%, 20% and 42% (P=0.02, P&amp;lt;0.001 and P&amp;lt;0.001, respectively versus untreated cells), whereas no effect was seen in MMR-proficient tumors (P=NS for all concentrations of olaparib). Finally, high-throughput profiling of hotspot mutations also more accurately detected microsatellite instability (MSI) in several cancer types compared to standard diagnostic methods. Sequencing of MMR-deficient tumors thus identified an unexpected synthetically lethal interaction between the major DNA repair pathways, offering novel treatment opportunities for MMR-deficient tumors. Citation Format: Betül T. Yesilyurt, Hui Zhao, Xavier Sagaert, Lieve Coenegrachts, Zeynep Kalender, Diego Garcia, Kim De Keersmaker, Gert Matthijs, Bernard Thienpont, Stein Aerts, Jan Cools, Frederic Amant, Diether Lambrechts. Sequencing of mismatch repair deficient tumors identifies a synthetic lethal interaction with other DNA repair pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2006. doi:10.1158/1538-7445.AM2013-2006

Resveratrol, but not dihydroresveratrol, induces premature senescence in primary human fibroblasts

Resveratrol, trans-3,5,4'-trihydroxystilbene, is a polyphenolic compound which has been reported to mimic the gene expression patterns seen in whole animals undergoing dietary restriction. The mechanism of action of resveratrol remains poorly understood, but modulation of both cellular proliferation and apoptosis has been proposed as important routes by which the molecule may exert its effects. This study reports the effects of both resveratrol and dihydroresveratrol (a primary in vivo metabolite) on the proliferative capacity of human primary fibroblasts. No generalised reduction in the growth fraction was observed when fibroblasts derived from three different tissues were treated with resveratrol at concentrations of 10 μm or less. However, concentrations above 25 μm produced a dose-dependent reduction in proliferation. This loss of the growth fraction was paralleled by an increase in the senescent fraction as determined by staining for senescence associated beta galactosidase and dose recovery studies conducted over a 7-day period. Entry into senescence in response to treatment with resveratrol could be blocked by a 30-min preincubation with the p38 MAP kinase inhibitor SB203580. No effects on proliferation were observed when cells were treated with dihydroresveratrol at concentrations of up to 100 μm.

Effect of raloxifene on human pancreatic adenocarcinoma growth in vitro and in vivo.

207 Background: The role of estrogen receptor (ER) signaling in pancreatic cancer is unknown. Recently, we demonstated that expression of the isoform ER beta correlates with an adverse prognosis in patients with pancreatic cancer. Here, we show that raloxifene, a specific estrogen receptor modulator (SERM), suppresses in vitro and in vivo tumor growth by interfering with ER beta signaling in human pancreatic adenocarcinoma. Methods: The human pancreatic adenocarcinoma cell line L3.6pl was cultured and exposed to raloxifene in vitro, and cell proliferation was determined by the BrdU assay. To analyze the specificity of raloxifene induced effects, ER knockdown was performed using siRNA specific for ER alpha and ER beta. In an in vivo model of orthotopic tumor xenografts in nude mice, raloxifene was administered daily, and tumor growth was monitored. Expression of ER beta and the proliferation marker Ki-67 were determined by immunohistochemistry. Results: Raloxifene treatment resulted in a potent, dose dependent reduction of proliferation in vitro over a nanomolar dose range. This effect was completely reversed by siRNA knockdown of ER beta, but not ER alpha, indicating an ER isotype specific signaling. In vivo, orthotopic tumor growth, as well as lymph node and liver metastases, was significantly suppressed in raloxifene treated mice. Analogous to the in vitro data, Ki-67 expression in vivo was significantly reduced in raloxifene treated mice, while ER beta expression was not changed in vivo. Conclusions: Inhibition of ER beta signaling by raloxifene results in a potent reduction of human pancreatic adenocarcinoma growth in vitro and in vivo. Treatment with SERMs may be an attractive therapeutic option in subjects expressing the ER beta isotype. No significant financial relationships to disclose.

Epigenetic Regulation of MicroRNA Expression In Hodgkin Lymphoma

AbstractAbstract 3885 Background:In recent years, microRNAs (miRNAs) have emerged as key regulators of carcinogenesis. miRNA expression is deregulated in multiple hematological malignancies. One of the mechanisms that can affect miRNA expression is methylation in the promoter regions of miRNA genes. The main objective of the present study was to identify tumor suppressor miRNAs that are silenced by alterations in gene methylation in Hodgkin Lymphoma (HL). In addition, since demethylating agents could affect miRNA expression, we evaluated the in vitro effectiveness of 5-Aza-2-deoxycytidine (AZA), a DNA methyltransferase inhibitor, in HL cell lines. Methods:To detect miRNAs regulated by methylation, we analyzed the expression of 670 mature miRNAs in two HL cell lines, L-428 and L-1236, before and after AZA treatment, by TaqMan Human MicroRNA Arrays V2.0 (Applied Biosystems) in an ABI 7900 HT sequence detection system. miRNA expression data was analyzed by the 2-ΔΔCt method using RNU48 as endogenous control. To validate the methylation status, genomic DNA samples from four HL cell lines (L-428, L-1236, HDMYZ and L-540) and peripheral B-cells from healthy donors were modified by sodium bisulfite using the EZ DNA Methylation kit (Zymo Research). The DNA methylation status of these samples was analyzed by methylation specific PCR (MSP) after sodium bisulfite modification of DNA. To evaluate AZA in vitro effectiveness, we treated the four HL cell lines daily with 20 nM, 250 nM, 1 μM and 5 μM of AZA or DMSO (vehicle control), and we assayed proliferation with CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) after 72 hours of treatment. Results:After AZA treatment in the L-428 and L-1236 HL cell lines, 32 miRNAs (of 670 analyzed) were expressed de novo, 15 of which were present in both cell lines. miR-34a, miR-203, miR-342, miR-105, miR-490, miR-375 and the miR-500 family had well-defined CpG islands in their promoter regions. The MSP analysis showed that miR-34a, miR-203, miR-490 and miR-375 were methylated in L-428 and L-1236 HL cell lines, but not in the normal B-cells. miR-342 was methylated in L-1236 HL cell line, but not in L-428 nor normal B-cells. To further validate this we analyzed the methylation pattern in HDMYZ and L-540 HL cell lines and found that only miR-203, miR-375 and miR-490 were methylated in all 4 HL cell lines analyzed. We observed an AZA dose-dependent reduction in proliferation in all four HL cell lines. After 72 hours of treatment with AZA at 5μM, proliferation significantly decreased by 29% in L-428, 36% in L-1236, 28% in HDMYZ and 22% in L-540. Moreover, the analysis of miRNA levels during the treatment showed a re-expression of methylated miRNAs after 48 hours. The methylation analysis of these miRNAs in laser captured microdisected HL cells from patients and functional analysis are ongoing and complete data will be reported. Conclusions:In summary, HL exhibits a characteristic epigenetic pattern which includes the methylation of key miRNAs during the carcinogenesis process, such as miR-34a and miR-203, which have previously been shown to act as tumor suppressors in lung, colorectal and other cancers. Moreover, we present evidence that demethylating agents allow the re-expression of these tumor suppressor miRNAs, suggesting that they could be a promising novel treatment for HL patients.Supported by FIS grant (PS09/00547). Disclosures:No relevant conflicts of interest to declare.

Different radiosensitivity of CD4+CD25+ regulatory T cells and effector T cells to low dose gamma irradiation in vitro

Purpose: To determine the radiosensitivity difference of human Cluster of Differentiation (CD)4+CD25+ regulatory T cells (Treg) and effector T cells to low dose gamma ray and elucidate the underlying mechanisms in vitro.Materials and methods: Blood samples were collected from five health subjects and five patients with advanced hepatocellular carcinoma (HCC). Treg and CD4+CD25− T cells were selected using magnetic microbeads. The proliferative profiles, cytokine secretion, and differential expressions of apoptosis-related proteins in Treg and CD4+CD25− T cells were compared using [3H]-thymidine incorporation, Luminex assay and flow cytometry when treated with various low doses of γ-ray.Results: A dose-dependent reduction of proliferation in response to irradiation which paralleled the induction of apoptosis existed in Treg and CD4+CD25− T cells. Treg were more radiosensitive to low-dose irradiation (0.94 Gray [Gy]) than effector T cells. The interferon-γ (IFNγ) was significantly upregulated and interleukin 10 (IL-10) was significantly downregulated in irradiated Treg. An enhanced immune response to low dose gamma ray existed in the peripheral blood in patients with advanced HCC. Higher levels of active caspase-3, CD95, B cell lymphoma 2 (Bcl-2)-associated X protein (Bax) expression were observed in Treg compared to CD4+CD25− T cells. In addition, gamma irradiation activated CD4+CD25− T cells to express CD25.Conclusions: These studies revealed that Treg were more radiosensitive than CD4+CD25− T cells to low dose irradiation. Higher expressions of apoptosis-related proteins such as caspase-3, CD95 and Bax were observed in Treg when compared to CD4+CD25− T cells. Our results suggest that treatment with low doses of gamma irradiation may be a viable strategy to enhance immune response in patients with advanced HCC.

A 28-Amino-Acid Peptide Fragment of the Cupredoxin Azurin Prevents Carcinogen-Induced Mouse Mammary Lesions

Azurin, a member of the cupredoxin family of redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and apoptotic effects. Azurin and amino acids 50-77 (p28) of azurin also produce a dose-dependent reduction in the proliferation of human mammary cancer by increasing the level of the tumor suppressor protein p53 in the cancer cell nucleus. We show that the development of 7,12-dimethylbenz[a]anthracene-induced hormone-dependent premalignant mammary ductal lesions and hormone-independent mammary alveolar lesions in mouse mammary gland organ culture is also significantly reduced by azurin and p28. The dose-dependent reduction in carcinogen-induced mammary cell proliferation by p28 was associated with an increase in the expression of p53. p28 also enhanced the inhibitory effect of a low dose of the antiestrogen tamoxifen on the development of hormone-dependent mammary ductal lesions, but did not enhance the inhibitory activity of fenretinide (N-4-hydroxyphenyl retinamide) on hormone-independent mammary alveolar lesions. These observations suggest that cupredoxins and fragments derived from them can exert a chemopreventive effect on carcinogen-induced mammary gland transformation, irrespective of hormonal environment, and enhance the inhibitory effects of tamoxifen in this model of preneoplastic mammary development.