Sp1 mRNA Research Articles

Abstract 3187: Nucleolin-induced cap-independent translational pathway involved in Sp1 accumulation in tumorigenesis.

Abstract Sp1 is a transcriptional factor to regulate numbers genes related to cell proliferation, apoptosis and tumor progression. Previous studies indicate that Sp1 is induced significantly through modulating its transcriptional activity and protein stability. To detail study the level of Sp1 protein and mRNA in tumorigenesis found that Sp1 mRNA was just increased a little, but Sp1 protein was increased dramatically, implying that enhancement of translational activity by nucleion might be important in the Sp1 accumulation in tumor progression. Here we found a Y-shift motif within the 5’-UTR of the Sp1 mRNA, and to study the contribution in Sp1 level found that the 5’-URT could enhance the translational efficiency of Sp1. Using this Y-shift motif as a probe fished the interacted proteins. Several RNA binding proteins were identified by LC/MS/MS, and nucleolin is one of them. To further address the effect of nucleolin in Sp1 induction found that nucleolin overexpression did not alter the level of Sp1 mRNA, but increased the translational activity obviously. In addition, the signal transduction pathway was also studied, and found that EGFR and Kras pathways might be involved in the induction in the cap-independent pathway in Sp1 induction in tumorigenesis. Taken the previous studies and this study together, during tumor formation, several strategies including increasing the transcriptional activity, protein stability and translational efficiency were used to induce the Sp1 level to regulate many oncogenes to enhance the cancer progression. Keywords: Nucleolin, Sp1, Cap-independent pathway, Citation Format: Tsung-I Hsu, Jan-Jong Hung, Wen-Chang Chang. Nucleolin-induced cap-independent translational pathway involved in Sp1 accumulation in tumorigenesis. [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 3187. doi:10.1158/1538-7445.AM2013-3187

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<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<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<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

Impaired caveolin‐1 contributes to hepatic ischemia and reperfusion injury via suppression of sphingosine kinase/sphingosine‐1‐phosphate receptor pathway

Caveolae are membrane structure enriched in glycosphingolipid and cholesterol and caveolin‐1 (Cav‐1) has been recognized to be pivotal in ischemic tolerance. Sphingosine‐1‐phosphate (S1P), one of the sphingolipid metabolites, is well known for its anti‐apoptotic properties to counteract against ischemia/reperfusion (I/R) injury and it acts through G protein‐coupled S1P receptors (S1P1‐S1P5). Recently, sphingosine kinases (SphK1 and SphK2) which phosphorylate sphingosine to S1P have been a target to regulate S1P pathway. Here, we investigated the cytoprotective role of Cav‐1 against I/R injury focusing on S1P signaling pathway. Mice underwent partial hepatic ischemia for 60 min followed by reperfusion. Recombinant peptides mimicking Cav‐1 scaffolding domain (CSD, 2.5 mg/kg, i.p.) was administered 1 h prior to ischemia. Ischemia itself reduced hepatic Cav‐1 protein level and this decrease persisted by 1 h of reperfusion. However, this decreased Cav‐1 protein level recovered to basal level 6 h of reperfusion and maintained by 24 h of reperfusion. The loss of caveolae structure and decreased Cav‐1 protein level was restored by CSD 1 h of reperfusion. Cav‐1 induction with CSD attenuated I/R‐induced increases in serum ALT and TNF‐α activity. The levels of SphK1 and Sphk2 mRNA decreased 1 h of reperfusion. CSD attenuated the decrease of SphK1 mRNA level while it did not affect SphK2. At 1 h of reperfusion, the levels of S1P1 and S1P2 mRNA decreased and CSD attenuated the decrease of S1P2 mRNA level while it did not affect S1P1. CSD also decreased phosphorylation of p38 MAPK and JNK. Moreover, I/R resulted in increases of cytochrome c release and apoptotic hepatocytes and CSD attenuated these increases. Our findings suggest that decreased Cav‐1 expression might contribute to hepatic I/R injury through down‐regulation of S1P pathway.

Resveratrol and Quercetin in Combination Have Anticancer Activity in Colon Cancer Cells and Repress Oncogenic microRNA-27a

Resveratrol and quercetin (RQ) in combination (1:1 ratio) previously inhibited growth in human leukemia cells. This study investigated the anticancer activity of the same mixture in HT-29 colon cancer cells. RQ decreased the generation of reactive oxygen species (ROS) by up to 2.25-fold and increased the antioxidant capacity by up to 3-fold in HT-29 cells (3.8–60 μg/mL), whereas IC50 values for viability were 18.13, 18.73, and 11.85 μg/mL, respectively. RQ also induced caspase-3-cleavage (2-fold) and increased PARP cleavage. Specificity protein (Sp) transcription factors are overexpressed in colon and other cancers and regulate genes required for cell proliferation survival and angiogenesis. RQ treatment decreased the expression of Sp1, Sp3, and Sp4 mRNA and this was accompanied by decreased protein expression. Moreover, the Sp-dependent antiapoptotic survival gene survivin was also significantly reduced, both at mRNA and protein levels. RQ decreased microRNA-27a (miR-27a) and induced zinc finger protein ZBTB10, an Sp-repressor, suggesting that interactions of RQ with the miR-27a-ZBTB10-axis play a role in Sp downregulation. This was confirmed by transfection of cells with the specific mimic for miR-27a, which partially reversed the effects of RQ. These findings are consistent with previous studies on botanical anticancer agents in colon cancer cells.

Regulation of integrin αV subunit expression by sulfatide in hepatocellular carcinoma cells

Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation.

Genome‐wide transcriptome analysis of the effects of sulforphane on normal and prostate cancer cells

Sulforaphane (SFN), a phytochemical derived from cruciferous vegetables, induces anti‐proliferative and pro‐apoptotic responses in prostate cancer cells, but not normal prostate cells. The mechanisms responsible for these specific chemopreventive properties remain unclear. We utilized RNA sequencing (RNA‐seq) to test the hypothesis that SFN modifies patterns of gene expression that are critical in prostate cancer progression. Normal prostate epithelial cells and prostate cancer cells were treated with 15 uM SFN and the transcriptome was determined at 6 and 24h time points. SFN alters the mRNA expression levels of ~3,000 genes in each sample and the transcriptional response is highly dynamic over time. SFN treatment also alters the expression of non‐coding RNAs including lincRNAs. SFN targets multiple pathways in normal and prostate cancer cells that are critical in cancer including proliferation, apoptosis, angiogenesis, DNA damage response, and cell migration. Network analysis suggests that the transcription factor Sp1 may be a major mediator by which SFN treatment induces changes in gene expression. Studies evaluating the effect of SFN on Sp1 are ongoing and Sp1 mRNA levels are significantly decreased by SFN treatment. Together, this information can be leveraged to help develop intervention strategies to significantly reduce the incidence and severity of prostate cancer. Funding: P01CA090890Grant Funding Source: National Institute of Health

Astaxanthin Suppresses MPP+-Induced Oxidative Damage in PC12 Cells through a Sp1/NR1 Signaling Pathway

Objective: To investigate astaxanthin (ATX) neuroprotection, and its mechanism, on a 1-methyl-4-phenyl-pyridine ion (MPP+)-induced cell model of Parkinson’s disease. Methods: Mature, differentiated PC12 cells treated with MPP+ were used as an in vitro cell model. The MTT assay was used to investigate cell viability after ATX treatment, and western blot analysis was used to observe Sp1 (activated transcription factor 1) and NR1 (NMDA receptor subunit 1) protein expression, real-time PCR was used to monitor Sp1 and NR1 mRNA, and cell immunofluorescence was used to determine the location of Sp1 and NR1 protein and the nuclear translocation of Sp1. Results: PC12 cell viability was significantly reduced by MPP+ treatment. The expression of Sp1 and NR1 mRNA and protein were increased compared with the control (p < 0.01). Following co-treatment with ATX and MPP+, cell viability was significantly increased, and Sp1 and NR1 mRNA and protein were decreased, compared with the MPP+ groups (p < 0.01). In addition, mithracycin A protected PC12 cells from oxidative stress caused by MPP+ by specifically inhibiting the expression of Sp1. Moreover, cell immunofluorescence revealed that ATX could suppress Sp1 nuclear transfer. Conclusion: ATX inhibited oxidative stress induced by MPP+ in PC12 cells, via the SP1/NR1 signaling pathway.

Analysis of Sp transcription factors in the postmortem brain of chronic schizophrenia: A pilot study of relationship to negative symptoms

Negative symptoms are the most resilient manifestations in schizophrenia. An imbalance in dopamine and glutamate pathways has been proposed for the emergence of these symptoms. SP1, SP3 and SP4 transcription factors regulate genes in these pathways, suggesting a possible involvement in negative symptoms. In this study, we characterized Sp factors in the brains of subjects with schizophrenia and explored a possible association with negative symptoms. We also included analysis of NR1, NR2A and DRD2 as Sp target genes. Postmortem cerebellum and prefrontal cortex from an antemortem clinically well-characterized and controlled collection of elderly subjects with chronic schizophrenia (n=16) and control individuals (n=14) were examined. We used the Positive and Negative Syndrome and the Clinical Global Impression Schizophrenia scales, quantitative PCR and immunoblot. SP1 protein and mRNA were reduced in the prefrontal cortex in schizophrenia whereas none of Sp factors were altered in the cerebellum. However, we found that SP1, SP3 and SP4 protein levels inversely correlated with negative symptoms in the cerebellum. Furthermore, NR2A and DRD2 mRNA levels correlated with negative symptoms in the cerebellum. In the prefrontal cortex, SP1 mRNA and NR1 and DRD2 inversely correlated with these symptoms while Sp protein levels did not. This pilot study not only reinforces the involvement of SP1 in schizophrenia, but also suggests that reduced levels or function of SP1, SP4 and SP3 may participate in negative symptoms, in part through the regulation of NMDA receptor subunits and/or Dopamine D2 receptor, providing novel information about the complex negative symptoms in this disorder.

Shugan-decoction relieves visceral hyperalgesia and reduces TRPV1 and SP colon expression

To evaluate the therapeutic effect of Shugan-decoction (SGD) on visceral hyperalgesia and colon gene expressions using a rat model. Ninety-six adult male Wistar rats were randomized into six equal groups for assessment of SGD effects on psychological stress-induced changes using the classic water avoidance stress (WAS) test. Untreated model rats were exposed to chronic (1 h/d for 10 d consecutive) WAS conditions; experimental treatment model rats were administered with intragastric SGD at 1 h before WAS on consecutive days 4-10 (low-dose: 0.1 g/mL; mid-dose: 0.2 g/mL; high-dose: 0.4 g/mL); control treatment model rats were similarly administered with the irritable bowel syndrome drug, dicetel (0.0042 g/mL); untreated normal control rats received no drug and were not subjected to the WAS test. At the end of the 10-d WAS testing period, a semi-quantitative measurement of visceral sensitivity was made by assessing the abdominal withdrawal reflex (AWR) to colorectal balloon-induced distension (at 5 mmHg increments) to determine the pain pressure threshold (PPT, evidenced by pain behavior). Subsequently, the animals were sacrificed and colonic tissues collected for assessment of changes in expressions of proteins related to visceral hypersensitivity (transient receptor potential vanilloid 1, TRPV1) and sustained visceral hyperalgesia (substance P, SP) by immunohistochemistry and real-time polymerase chain reaction. Inter-group differences were assessed by paired t test or repeated measures analysis of variance. The WAS test successfully induced visceral hypersensitivity, as evidenced by a significantly reduced AWR pressure in the untreated model group as compared to the untreated normal control group (190.4 ± 3.48 mmHg vs 224.0 ± 4.99 mmHg, P < 0.001). SGD treatments at mid-dose and high-dose and the dicetel treatment significantly increased the WAS-reduced PPT (212.5 ± 2.54, 216.5 ± 3.50 and 217.7 ± 2.83 mmHg respectively, all P < 0.001); however, the low-dose SGD treatment produced no significant effect on the WAS-reduced PPT (198.3 ± 1.78 mmHg, P > 0.05). These trends corresponded to the differential expressions observed for both TRPV1 protein (mid-dose: 1.64 ± 0.08 and high-dose: 1.69 ± 0.12 vs untreated model: 3.65 ± 0.32, P < 0.001) and mRNA (0.44 ± 0.16 and 0.15 ± 0.03 vs 1.39 ± 0.15, P < 0.001) and SP protein (0.99 ± 0.20 and 1.03 ± 0.23 vs 2.03 ± 0.12, P < 0.01) and mRNA (1.64 ± 0.19 and 1.32 ± 0.14 vs 2.60 ± 0.33, P < 0.05). These differential expressions of TRPV1 and SP related to mid- and high-dose SGD treatments were statistically similar to the changes induced by dicetel treatment. No signs of overt damage to the rat system were observed for any of the SGD dosages. Shugan-decoction can reduce chronic stress-induced visceral hypersensitivity in rats, and the regulatory mechanism may involve mediating the expressions of TRPV1 and SP in colon tissues.

Synergistic Effect of Caffeine and Glucocorticoids on Expression of Surfactant Protein B (SP-B) mRNA

Administration of glucocorticoids and caffeine is a common therapeutic intervention in the neonatal period, but possible interactions between these substances are still unclear. The present study investigated the effect of caffeine and different glucocorticoids on expression of surfactant protein (SP)-B, crucial for the physiological function of pulmonary surfactant. We measured expression levels of SP-B, various SP-B transcription factors including erythroblastic leukemia viral oncogene homolog 4 (ErbB4) and thyroid transcription factor-1 (TTF-1), as well as the glucocorticoid receptor (GR) after administering different doses of glucocorticoids, caffeine, cAMP, or the phosphodiesterase-4 inhibitor rolipram in the human airway epithelial cell line NCI-H441. Administration of dexamethasone (1 µM) or caffeine (5 mM) stimulated SP-B mRNA expression with a maximal of 38.8±11.1-fold and 5.2±1.4-fold increase, respectively. Synergistic induction was achieved after co-administration of dexamethasone (1 mM) in combination with caffeine (10 mM) (206±59.7-fold increase, p<0.0001) or cAMP (1 mM) (213±111-fold increase, p = 0.0108). SP-B mRNA was synergistically induced also by administration of caffeine with hydrocortisone (87.9±39.0), prednisolone (154±66.8), and betamethasone (123±6.4). Rolipram also induced SP-B mRNA (64.9±21.0-fold increase). We detected a higher expression of ErbB4 and GR mRNA (7.0- and 1.7-fold increase, respectively), whereas TTF-1, Jun B, c-Jun, SP1, SP3, and HNF-3α mRNA expression was predominantly unchanged. In accordance with mRNA data, mature SP-B was induced significantly by dexamethasone with caffeine (13.8±9.0-fold increase, p = 0.0134). We found a synergistic upregulation of SP-B mRNA expression induced by co-administration of various glucocorticoids and caffeine, achieved by accumulation of intracellular cAMP. This effect was mediated by a caffeine-dependent phosphodiesterase inhibition and by upregulation of both ErbB4 and the GR. These results suggested that caffeine is able to induce the expression of SP-transcription factors and affects the signaling pathways of glucocorticoids, amplifying their effects. Co-administration of caffeine and corticosteroids may therefore be of benefit in surfactant homeostasis.

Sphingosine 1-Phosphate (S1P) Carrier-dependent Regulation of Endothelial Barrier: HIGH DENSITY LIPOPROTEIN (HDL)-S1P PROLONGS ENDOTHELIAL BARRIER ENHANCEMENT AS COMPARED WITH ALBUMIN-S1P VIA EFFECTS ON LEVELS, TRAFFICKING, AND SIGNALING OF S1P1

Sphingosine 1-phosphate (S1P) is a blood-borne lysosphingolipid that acts to promote endothelial cell (EC) barrier function. In plasma, S1P is associated with both high density lipoproteins (HDL) and albumin, but it is not known whether the carriers impart different effects on S1P signaling. Here we establish that HDL-S1P sustains EC barrier longer than albumin-S1P. We showed that the sustained barrier effects of HDL-S1P are dependent on signaling by the S1P receptor, S1P1, and involve persistent activation of Akt and endothelial NOS (eNOS), as well as activity of the downstream NO target, soluble guanylate cyclase (sGC). Total S1P1 protein levels were found to be higher in response to HDL-S1P treatment as compared with albumin-S1P, and this effect was not associated with increased S1P1 mRNA or dependent on de novo protein synthesis. Several pieces of evidence indicate that long term EC barrier enhancement activity of HDL-S1P is due to specific effects on S1P1 trafficking. First, the rate of S1P1 degradation, which is proteasome-mediated, was slower in HDL-S1P-treated cells as compared with cells treated with albumin-S1P. Second, the long term barrier-promoting effects of HDL-S1P were abrogated by treatment with the recycling blocker, monensin. Finally, cell surface levels of S1P1 and levels of S1P1 in caveolin-enriched microdomains were higher after treatment with HDL-S1P as compared with albumin-S1P. Together, the findings reveal S1P carrier-specific effects on S1P1 and point to HDL as the physiological mediator of sustained S1P1-PI3K-Akt-eNOS-sGC-dependent EC barrier function.

MiR-29b Exerts Anti-Multiple Myeloma Activity by Targeting Key Oncogenic Pathways and Modulating DNA Methylation Profile.

Abstract Abstract 2941 The enforced expression of tumor suppressor microRNAs (miRNAs) is a promising strategy for cancer treatment. Among miRNAs frequently deregulated in cancer, miR-29b is of great interest since its overexpression leads to tumor growth inhibition in solid tumors and leukemias. On these bases, we first investigated miR-29b expression pattern in multiple myeloma (MM) cells. miRNA profiling of primary CD138+ MM patient cells (n=55) revealed deregulated expression of miR-29b with almost 60% of cases showing strongly reduced levels. Moreover, as assessed by quantitative real time PCR (qRT-PCR), miR-29b expression was significantly downregulated in a panel of 11 MM cell lines and even further decreased when RPMI-8226 MM cells were co-cultured with bone marrow stromal cells (BMSCs), indicating that the human bone marrow microenvironment (huBMM) modulates miR-29b levels. Either transient expression of synthetic miR-29b mimics through electroporation or stable lentivirus-enforced miR-29b expression induced growth inhibition and apoptosis of MM cells in vitro. In vivo intratumoral or systemic delivery of neutral lipidic formulated-miR-29b mimics in different clinically relevant murine models of human MM, including our recent SCID-synth hu model which recapitulates the huBMM, led to significant tumor growth inhibition together with increased survival of miR-29b-treated animals. By Western blot and qRT-PCR analysis, we observed dramatic reduction of predicted targets of miR-29b including CDK6 and MCL-1, which can thus explain antiproliferative and pro-apoptotic effects triggered by miR-29b expression. Most importantly, we identified Sp1, a transcription factor endowed with oncogenic activity in MM, as a negative regulator of miR-29b expression providing evidence of a novel regulatory loop in MM cells: in fact, enforced expression of Sp1 was able to reduce miR-29b-promoter activity and miR-29b levels, whereas miR-29b expression decreased Sp1 mRNA and protein levels via 3'UTR binding, as assessed in luciferase reporter assays. Moreover, treatment of MM cells with the proteasome inhibitor bortezomib led to Sp1 downregulation and miR-29b upregulation. Strikingly, miR-29b transfection significantly strengthened the in vitro anti-proliferative and apoptotic effects induced by bortezomib, thus highlighting its role in the mechanism of anti-MM activity of this drug. Additional miR-29b targets include de novo DNA methyltransferase 3A (DNMT3A) and DNA methyltransferase 3B (DNMT3B). Microarray profiling revealed increased DNMT3A and DNMT3B mRNA levels in cancer cells as compared to normal plasma cells. The integrated analysis of miRNA/mRNA profiling in a panel of 18 MM cell lines highlighted an inverse correlation between miR-29b and DNMT3B levels. Finally, miR-29b was proven to target DNMT3A and DNMT3B and significantly reduced the global DNA methylation levels in MM cells, as assessed by performing an in vitro DNA methylation assay with genomic DNA extracted from MM cells after synthetic miR-29b electroporation. In conclusion, our findings indicate that miR-29b exerts anti-MM activity by targeting relevant oncogenic pathways and by restoring the aberrant methylation pattern of MM cells. These results support a potential therapeutic role for miR-29b mimics in MM as single agent or in combination with the proteasome inhibitor bortezomib and/or demethylating agents. Disclosures: Munshi: Celgene: Consultancy; Millenium: Consultancy; Merck: Consultancy; Onyx: Consultancy.

MicroRNA-29b contributes to DNA hypomethylation of CD4+ T cells in systemic lupus erythematosus by indirectly targeting DNA methyltransferase 1

The mechanism of DNA hypomethylation in systemic lupus erythematosus (SLE) has not been fully elucidated. Recent studies showed that miR-29b could regulate DNA methylation by targeting the DNA methylation machinery. However, the role of miR-29b in T cell aberrant DNA hypomethylation of SLE still remains unclear. In this study, we asked whether miR-29b regulate DNA methylation in lupus CD4+ T cells. The miR-29b expression was analyzed by quantitative polymerase chain reaction (qPCR). Sp1, DNMT1, CD11a and CD70 mRNA and protein levels were determined by qPCR, Western-blotting and flow cytometry, respectively. The global DNA methylation levels were evaluated by the Methyflash™ DNA Methylation Quantification Kit. CD11a and CD70 promoter methyaltion levels were detected by bisulfate modification and methylation-sensitive high resolution melting analysis. In SLE patients, the miR-29b levels were up-regulated as compared to healthy donors and its degree of overexpression was negatively correlated with sp1 and DNMT1 protein levels, respectively. Overexpression of miR-29b resulted in significant reduction of sp1 and DNMT1 expression. Further analysis demonstrated that overexpression of miR-29b in CD4+ T cells from healthy donors led to the DNA hypomethylation and up-regulation of genes encoding CD11a and CD70, and inhibition of miR-29b expression in CD4+ T cells from patients with lupus caused reverse effects. Our study suggests that miR-29b negatively regulates DNMT1 expression by targeting sp1 in T cells. The overexpression of miR-29b contributes to the reduction of DNMT1 levels and thereby DNA hypomethylation in SLE. This finding provides potential novel strategies for therapeutic interventions.

Identification of novel Sp1 targets involved in proliferation and cancer by functional genomics

Sp1 is a transcription factor regulating many genes through its DNA binding domain, containing three zinc fingers. We were interested in identifying target genes regulated by Sp1, particularly those involved in proliferation and cancer. Our approach was to treat HeLa cells with a siRNA directed against Sp1 mRNA to decrease the expression of Sp1 and, in turn, the genes activated by this transcription factor. Sp1-siRNA treatment led to a great number of differentially expressed genes as determined by whole genome cDNA microarray analysis. Underexpressed genes were selected since they represent putative genes activated by Sp1 and classified in six Gene Onthology categories, namely proliferation and cancer, mRNA processing, lipid metabolism, glucidic metabolism, transcription and translation. Putative Sp1 binding sites were found in the promoters of the selected genes using the Match™ software. After literature mining, 11 genes were selected for further validation. Underexpression by qRT-PCR was confirmed for the 11 genes plus Sp1 in HeLa cells after Sp1-siRNA treatment. EMSA and ChIP assays were performed to test for binding of Sp1 to the promoters of these genes. We observed binding of Sp1 to the promoters of RAB20, FGF21, IHPK2, ARHGAP18, NPM3, SRSF7, CALM3, PGD and Sp1 itself. Furthermore, the mRNA levels of RAB20, FGF21 and IHPK2 and luciferase activity for these three genes related to proliferation and cancer, were determined after overexpression of Sp1 in HeLa cells, to confirm their regulation by Sp1. Involvement of these three genes in proliferation was validated by gene silencing using polypurine reverse hoogsteen hairpins.

IRF1 suppresses Ki-67 promoter activity through interfering with Sp1 activation

Interferon regulatory factor 1 (IRF1) shows tumor-suppressor activity by suppressing proliferation of cancer cells. To exert its anti-proliferative effects, this factor must ultimately control transcription of several key genes that regulate cell cycle progression. Here, we showed that Ki-67 gene is a novel proliferation-related downstream target of IRF1. IRF1 repressed Ki-67 gene transcription in a dose-dependent manner in human Ketr-3 and 786-O renal carcinoma cells. We previously cloned the Ki-67 core promoter which contained two functional Sp1 binding sites. Mutation of the two Sp1 binding sites abrogated Sp1-dependent enhancement of Ki-67 promoter activity. Forced elevation of IRF1 decreased endogenous Sp1 protein level. However, there was no effect on Sp1 mRNA level after transfected with IRF1. Our findings establish a casual series of events that connect anti-proliferative effects of IRF1 with the Ki-67 gene, which encodes a key regulator of the G1/S phase transition. It suggests that the inhibitory effect on Ki-67 gene expression mediated by decreasing level of Sp1 protein might be a novel function of the anti-tumor activity of IRF1.

Natural diterpenes from coffee, cafestol and kahweol induce apoptosis through regulation of specificity protein 1 expression in human malignant pleural mesothelioma

BackgroundMalignant pleural mesothelioma (MPM) is a highly aggressive cancer with a very poor prognosis. Several clinical studies such as immunotherapy, gene therapy and molecular targeting agents have been tried for treatment of malignant mesothelioma, however, there is no application for effective clinical treatment. Coffee has various biological functions such as anti-oxidant, anti-inflammatory, anti-mutagenic and anti-carcinogenic activities. The therapeutic activities of the bioactive compounds in coffee was sugested to influence intracellular signaling of MPM. Regarding to the cancer-related functions, In this study, suppression of Sp1 protein level followed by induction of MSTO-211H cell apoptosis by cafestol and kahweol were investigated in oreder to determine Sp1's potential as a significant target for human MPM therapy as well.MethodsCells were treated separately with final concentration of cafestol and kahweol and the results were analyzed by MTS assay, DAPI staining, PI staining, luciferase assay, RT-PCR, and immunoblotting.ResultsViability of MSTO-211H and H28 cells were decreased, and apoptotic cell death was increased in MSTO-211H as a result of cafestol and kahweol treatment. Cafestol and kahweol increased Sub-G1 population and nuclear condensation in MSTO-211H cells. Roles of Sp1 in cell proliferation and apoptosis of the MSTO-211H cells by the Sp1 inhibitor of Mithramycin A were previously confirmed. Cafestol and kahweol significantly suppressed Sp1 protein levels. Kahweol slightly attenuated Sp1 mRNA, while Cafestol did not affect in MSTO-211H cells. Cafestol and kahweol modulated the promoter activity and protein expression level of the Sp1 regulatory genes including Cyclin D1, Mcl-1, and Survivin in mesothelioma cells. Apoptosis signaling cascade was activated by cleavages of Bid, Caspase-3, and PARP with cafestol and by upregulation of Bax, and downregulation of Bcl-xl by kahweol.ConclusionsSp1 can be a novel molecular target of cafestol and kahweol in human MPM.

The Role of Sphingosine Kinase 1 in Patients With Severe Acute Pancreatitis

To investigate the role of sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) signaling in inflammatory response in severe acute pancreatitis (SAP). SAP is an acute inflammatory process of the pancreas, which may lead to systemic inflammatory response syndrome and multiorgan dysfunction syndrome. SphK1 and its product S1P have been implicated in inflammatory response and various immune cell functions. However, the potential role for SphK1/S1P in inflammatory response in SAP is still unclear. Twenty-two patients with SAP were enrolled in this study. SphK1 expression on peripheral neutrophils, monocytes, and lymphocytes was evaluated by flow cytometry. SphK enzymatic activity in neutrophils and lymphocytes was measured using a radiometric assay. The expression of S1P1 and S1P3 mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The serum levels of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1β), and IL-6 were measured by ELISA. The expression of SphK1 and SphK activity were markedly increased in peripheral immune cells in the early stage of SAP and then reduced in the restoration stage in the patients. Moreover, we found that the level of S1P3 mRNA in peripheral neutrophils and lymphocytes of SAP patients was significantly elevated in the early stage as compared with the healthy volunteers, and it reduced in the restoration period. SphK1 expression on human peripheral neutrophils, monocytes, and CD4(+) T lymphocytes were positively correlated with the APACHE (Acute Physiological and Chronic Health Evaluation) II scores in patients with SAP. The levels of serum proinflammatory cytokines including TNF-α, IL-1β, and IL-6 showed similar shifts with intracellular SphK1 expression in SAP patients. The authors identified a link between the SphK1 expression on peripheral immune cells and the severity of SAP. Observations showed a possible immunomodulating role for SphK1/S1P signaling in inflammatory response in SAP, suggesting that regulation of SphK1/S1P pathway may represent novel targets in the treatment of SAP.

The flavonoid resveratrol suppresses growth of human malignant pleural mesothelioma cells through direct inhibition of specificity protein 1

Resveratrol (Res), from the skin of red grapes, induces apoptosis in some malignant cells, but there are no reports on the apoptotic effect of Res on human malignant pleural mesothelioma. We found that Res interacts with specificity protein 1 (Sp1). The IC50 for Res was 17 µM in MSTO-211H cells. Cell viability was decreased and apoptotic cell death was increased by Res (0-60 µM). Res increased the Sub-G1 population in MSTO-211H cells and significantly suppressed Sp1 protein levels, but not Sp1 mRNA levels. Res modulated the expression of Sp1 regulatory proteins including p21, p27, cyclin D1, Mcl-1 and survivin in mesothelioma cells. After treatment with Res, apoptosis signaling cascades were activated by the activation of Bid, Bim, caspase-3 and PARP, upregulation of Bax and downregulation of Bcl-xL. Res (20 mg/kg daily for 4 weeks) effectively suppressed tumor growth in vivo in BALB/c athymic (nu+/nu+) mice injected with MSTO-211H cells, an effect that was mediated by inhibition of Sp1 expression and induction of apoptotic cell death. Our results strongly suggest that Sp1 is a novel molecular target of Res in human malignant pleural mesothelioma.

Betulinic acid decreases ER‐negative breast cancer cell growth in vitro and in vivo: Role of Sp transcription factors and microRNA‐27a:ZBTB10

Betulinic acid (BA), a pentacyclic triterpenoid isolated from tree bark is cytotoxic to cancer cells. There is evidence that specificity proteins (Sps), such as Sp1, Sp3, and Sp4, are overexpressed in tumors and contribute to the proliferative and angiogenic phenotype associated with cancer cells. The objective of this study was to determine the efficacy of BA in decreasing the Sps expression and underlying mechanisms. Results show that BA decreased proliferation and induced apoptosis of estrogen-receptor-negative breast cancer MDA-MB-231 cells. The BA-induced Sp1, Sp3, and Sp4 downregulation was accompanied by increased zinc finger ZBTB10 expression, a putative Sp-repressor and decreased microRNA-27a levels, a microRNA involved in the regulation of ZBTB10. Similar results were observed in MDA-MB-231 cells transfected with ZBTB10 expression plasmid. BA induced cell cycle arrest in the G2/M phase and increased Myt-1 mRNA (a microRNA-27a target gene), which causes inhibition in G2/M by phosphorylation of cdc2. The effects of BA were reversed by transient transfection with a mimic of microRNA-27a. In nude mice with xenografted MDA-MB-231 cells, tumor size and weight were significantly decreased by BA treatment. In tumor tissue, ZBTB10 mRNA was increased while mRNA and protein of Sp1, Sp3 and Sp4, as well as mRNA of vascular endothelial growth factor receptor (VEGFR), survivin and microRNA-27a were decreased by BA. In lungs of xenografted mice, human β2-microglobulin mRNA was decreased in BA-treated animals. These results show that the anticancer effects of BA are at least in part based on interactions with the microRNA-27a-ZBTB10-Sp-axis causing increased cell death.

Sp1 and VEGF mRNA expression in the retina micrangium endothelial cells of streptozotocin-induced diabetic rats

The aim of this study is to evaluate the Sp1 and VEGF mRNA expression in the retina micrangium endothelial cells of streptozotocin-induced diabetic rats. At the end of our study, we determined that SP1 positive expression rate and the Sp1, VEGF mRNA and protein expression in the retina micrangium endothelial cells in isolated rat eyes of diabetic rats were significantly increased compared to normal animals. The ERK, Sp1 and VEGF protein expression in the retina micrangium endothelial cells of diabetic rats was significantly decreased in high-surcose (Sp1 specific inhibitor Mithramycin A or ERK specific inhibitor PD98059) culture group in comparison to normal (vechile PBS) culture group. These findings suggest that ERK, Sp1 and VEGF protein expression is an important factor to affect the occurrence and development of vision diseases in diabetic patients. Key words: Sp1, VEGF, PCR, Retina, Immunohistochemistry