HOTAIR is a long intervening non-coding RNA (lincRNA) that associates with the Polycomb Repressive Complex 2 (PRC2) and overexpression is correlated with poor survival for breast, colon and liver cancer patients. In this study, we show that HOTAIR expression is increased in pancreatic tumors compared to non-tumor tissue and is associated with more aggressive tumors. Knockdown of HOTAIR (siHOTAIR) by RNA interference shows that HOTAIR plays an important role in pancreatic cancer cell invasion and as reported in other cancer cell lines. In contrast, HOTAIR knockdown in Panc1 and L3.6pL pancreatic cancer cells that overexpress this lincRNA decreased cell proliferation, altered cell cycle progression, and induced apoptosis, demonstrating an expanded function for HOTAIR in pancreatic cancer cells compared to other cancer cell lines. Results of gene array studies showed that there was minimal overlap between HOTAIR-regulated genes in pancreatic vs. breast cancer cells and HOTAIR uniquely suppressed several interferon-related genes and gene sets related to cell cycle progression in pancreatic cancer cells and tumors. Analysis of selected genes suppressed by HOTAIR in Panc1 and L3.6 pL cells showed by knockdown of EZH2 and chromatin immunoprecipitation assays that HOTAIR-mediated gene repression was both PRC2-dependent and -independent. HOTAIR knockdown in L3.6pL cells inhibited tumor growth in mouse xenograft model, further demonstrating the pro-oncogenic function of HOTAIR in pancreatic cancer.

Curcumin activates diverse anticancer activities that lead to inhibition of cancer cell and tumor growth, induction of apoptosis, and antiangiogenic responses. In this study, we observed that curcumin inhibits Panc28 and L3.6pL pancreatic cancer cell and tumor growth in nude mice bearing L3.6pL cells as xenografts. In addition, curcumin decreased expression of p50 and p65 proteins and NFB-dependent transactivation and also decreased Sp1, Sp3, and Sp4 transcription factors that are overexpressed in pancreatic cancer cells. Because both Sp transcription factors and NFB regulate several common genes such as cyclin D1, survivin, and vascular endothelial growth factor that contribute to the cancer phenotype, we also investigated interactions between Sp and NFB transcription factors. Results of Sp1, Sp3, and Sp4 knockdown by RNA interference demonstrate that both p50 and p65 are Sp-regulated genes and that inhibition of constitutive or tumor necrosis factor-induced NFB by curcumin is dependent on down-regulation of Sp1, Sp3, and Sp4 proteins by this compound. Curcumin also decreased mitochondrial membrane potential and induced reactive oxygen species in pancreatic cancer cells, and this pathway is required for down-regulation of Sp proteins in these cells, demonstrating that the mitochondriotoxic effects of curcumin are important for its anticancer activities.

Curcumin is the active component of tumeric, and this polyphenolic compound has been extensively investigated as an anticancer drug that modulates multiple pathways and genes. In this study, 10 to 25 Mmol/L curcumin inhibited 253JB-V and KU7 bladder cancer cell growth, and this was accompanied by induction of apoptosis and decreased expression of the proapoptotic protein survivin and the angiogenic proteins vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1). Because expression of survivin, VEGF, and VEGFR1 are dependent on specificity protein (Sp) transcription factors, we also investigated the effects of curcumin on Sp protein expression as an underlying mechanism for the apoptotic and antiangiogenic activity of this compound. The results show that curcumin induced proteasome-dependent down-regulation of Sp1, Sp3, and Sp4 in 253JB-V and KU7 cells. Moreover, using RNA interference with small inhibitory RNAs for Sp1, Sp3, and Sp4, we observed that curcumin-dependent inhibition of nuclear factor KB (NF-KB)-dependent genes, such as bcl-2, survivin, and cyclin D1, was also due, in part, to loss of Sp proteins. Curcumin also decreased bladder tumor growth in athymic nude mice bearing KU7 cells as xenografts and this was accompanied by decreased Sp1, Sp3, and Sp4 protein levels in tumors. These results show for the first time that one of the underlying mechanisms of action of curcumin as a cancer chemotherapeutic agent is due, in part, to decreased expression of Sp transcription factors in bladder cancer cells. [Cancer Res 2008;68(13):5345-54]

The anticancer agent 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its methyl ester (CDDO-Me) typically induce a broad spectrum of growth-inhibitory, proapoptotic, and antiangiogenic responses. Treatment of Panc1, Panc28, and L3.6pL pancreatic cancer cells with low micromolar concentrations of CDDO or CDDO-Me resulted in growth inhibition, induction of apoptosis, and down-regulation of cyclin D1, survivin, vascular endothelial growth factor (VEGF), and its receptor (VEGFR2). RNA interference studies indicate that these repressed genes are regulated by specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and Western blot analysis of lysates from pancreatic cancer cells treated with CDDO and CDDO-Me shows for the first time that both compounds decreased the expression of Sp1, Sp3, and Sp4. Moreover, CDDO-Me (7.5 mg/kg/day) also inhibited pancreatic human L3.6pL tumor growth and down-regulated Sp1, Sp3, and Sp4 in tumors using an orthotopic pancreatic cancer model. CDDO-Me also induced reactive oxygen species (ROS) and decreased mitochondrial membrane potential (MMP) in Panc1 and L3.6pL cells, and cotreatment with antioxidants (glutathione and dithiothreitol) blocked the formation of ROS, reversed the loss of MMP, and inhibited down-regulation of Sp1, Sp3, and Sp4. Repression of Sp and Sp-dependent genes by CDDO-Me was due to the down-regulation of microRNA-27a and induction of zinc finger and BTB domain containing 10 (ZBTB10), an Sp repressor, and these responses were also reversed by antioxidants. Thus, the anticancer activity of CDDO-Me is due, in part, to activation of ROS, which in turn targets the microRNA-27a:ZBTB10-Sp transcription factor axis. This results in decreased expression of Spregulated genes, growth inhibition, induction of apoptosis, and antiangiogenic responses.Extracts of plants and microorganisms and individual natural products have been extensively used as traditional medicines for the treatment of several diseases, including cancer.Individual natural products including aspirin, morphine, quinine, statins, penicillins, taxanes, and many other compounds are widely used pharmaceutical agents and serve as templates for the synthesis of more potent analogs (Koehn and Carter, 2005). Triterpenoids are derived from cyclization of oxidosqualene, and different cyclization pathways coupled with postcyclization modifications can give several thousand possible analogs, including oleanolic acid, which contains a pentacyclic oleanane skeleton and a C28 carboxyl group. Oleanolic acid has been used by Sporn, Honda, and their collaborators as a template for extensive structure-activity ABBREVIATIONS: CDDO, 2-cyano-3,12-dioxoleana-1,9-dien-28-oic acid; CDDO-IM, imidazole ester of 2-cyano-3,12-dioxoleana-1,9-dien-28-oic acid; CDDO-Me, methyl ester of 2-cyano-3,12-dioxoleana-1,9-dien-28-oic acid; CDODA, 2-cyano-3,11-dioxo-18␤-olean-1,12-dien-30-oic acid; CDODA-Me, methyl ester of 2-cyano-3,11-dioxo-18␤-olean-1,12-dien-30-oic acid; GSH, glutathione; MMP, mitochondrial membrane pote...

Methyl 2-cyano-3,11-dioxo-18b-olean-1,12-dien-30-oate (CDODAMe) is a synthetic derivative of glycyrrhetinic acid, a triterpenoid phytochemical found in licorice extracts. CDODA-Me inhibited growth of RKO and SW480 colon cancer cells and this was accompanied by decreased expression of Sp1, Sp3 and Sp4 protein and mRNA and several Sp-dependent genes including survivin, vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1 or Flt-1). CDODA-Me also induced apoptosis, arrested RKO and SW480 cells at G 2 /M, and inhibited tumor growth in athymic nude mice bearing RKO cells as xenografts. CDODA-Me decreased expression of microRNA-27a (miR-27a), and this was accompanied by increased expression of 2 miR-27a-regulated mRNAs, namely ZBTB10 (an Sp repressor) and Myt-1 which catalyzes phosphorylation of cdc2 to inhibit progression of cells through G 2 /M. Both CDODA-Me and antisense miR-27a induced comparable responses in RKO and SW480 cells, suggesting that the potent anticarcinogenic activity of CDODA-Me is due to repression of oncogenic miR-27a. ' UICCKey words: CDODA-Me; anticarcinogenicity; miR-27a; colon cancer; cell cycle MicroRNAs (miRNAs) are 20-25 bp oligonucleotides that interact with complementary binding sites in 3 0 -untranslated regions of target mRNAs to inhibit their expression by blocking translation or by decreasing mRNA stability.1,2 miRNA interactions with mRNA requires the overlap of 6-8 base pairs and, due to this relatively low stringency, computational studies show that miRNAs can potentially interact with several hundred mRNAs.Despite this lack of specificity, miRNAs have a profound effect on gene expression and cellular homeostasis and, in cancer cells, expression of several critical oncogenes and tumor suppressor genes are regulated by miRNA expression.3-6 miR-221 and miR-222 target the cyclin-dependent kinase inhibitor p27 6 and miR-21 decreases expression of several mRNAs including the tumor suppressor gene tropomyosin 1. 3 Recent studies in this laboratory showed that miR-27a targets ZBTB10 mRNA, a putative zinc finger protein that suppresses specificity protein (Sp) transcription factors and Sp-dependent gene expression. 7 The Sp transcription factors Sp1, Sp3 and Sp4 are highly expressed in cancer cell lines, and results of RNA interference studies show that Sp proteins regulate expression of angiogenic genes such as vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR1, Flt-1), VEGFR2 (KDR) and the antiapoptotic gene survivin. 8-14Betulinic acid and the nonsteroidal anti-inflammatory drug tolfenamic acid inhibit prostate and pancreatic cell and tumor growth through activation of proteasome-dependent degradation of Sp1, Sp3 and Sp4 proteins. 13,14 In our study, we show that methyl 2-cyano-3,11-dioxo-18b-olean-1,12-dien-30-oate (CDODA-Me) is highly cytotoxic to colon cancer cells and also decreases Sp and Sp-dependent genes and proteins. However, these effects are proteasome-independent. We now show for the first time that CDODA-Me acts through downregulation of...