Maen Abdelrahim

The Aryl Hydrocarbon Receptor Mediates Degradation of Estrogen Receptor α through Activation of Proteasomes

ABSTRACT 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands suppress 17β-estradiol (E)-induced responses in the rodent uterus and mammary tumors and in human breast cancer cells. Treatment of ZR-75, T47D, and MCF-7 human breast cancer cells with TCDD induces proteasome-dependent degradation of endogenous estrogen receptor α (ERα). The proteasome inhibitors MG132, PSI, and PSII inhibit the proteasome-dependent effects induced by TCDD, whereas the protease inhibitors EST, calpain inhibitor II, and chloroquine do not affect this response. ERα levels in the mouse uterus and breast cancer cells were significantly lower after cotreatment with E plus TCDD than after treatment with E or TCDD alone, and our results indicate that AhR-mediated inhibition of E-induced transactivation is mainly due to limiting levels of ERα in cells cotreated with E plus TCDD. TCDD alone or in combination with E increases formation of ubiquitinated forms of ERα, and both coimmunoprecipitation and mammalian two-hybrid assays demonstrate that TCDD induces interaction of the AhR with ERα in the presence or absence of E. In contrast, E does not induce AhR-ERα interactions. Thus, inhibitory AhR-ERα cross talk is linked to a novel pathway for degradation of ERα in which TCDD initially induces formation of a nuclear AhR complex which coordinately recruits ERα and the proteasome complex, resulting in degradation of both receptors.

Role of Sp Proteins in Regulation of Vascular Endothelial Growth Factor Expression and Proliferation of Pancreatic Cancer Cells

Sp proteins play an important role in angiogenesis and growth of cancer cells, and specificity protein 1 (Sp1) has been linked to vascular endothelial growth factor (VEGF) expression in pancreatic cancer cells. RNA interference was used to investigate the role of Sp family proteins on regulation of VEGF expression and proliferation of Panc-1 pancreatic cancer cells. Using a series of constructs containing VEGF promoter inserts, it was initially shown that Sp1 and Sp3 were required for transactivation, and this was primarily dependent on proximal GC-rich motifs. We also showed that Sp4 was expressed in Panc-1 cells, and RNA interference assays suggested that Sp4 cooperatively interacted with Sp1 and Sp3 to activate VEGF promoter constructs in these cells. However, the relative contributions of Sp proteins to VEGF expression were variable among different pancreatic cancer cell lines. Small inhibitory RNAs for Sp3, but not Sp1 or Sp4, inhibited phosphorylation of retinoblastoma protein, blocked G0/G1 → S-phase progression, and up-regulated p27 protein/promoter activity of Panc-1 cells; similar results were observed in other pancreatic cancer cells, suggesting that Sp3-dependent growth of pancreatic cancer cells is caused by inhibition of p27 expression.

Estrogen regulation of vascular endothelial growth factor gene expression in ZR-75 breast cancer cells through interaction of estrogen receptor |[alpha]| and SP proteins

Vascular endothelial growth factor (VEGF) is expressed in multiple hormone-dependent cancer cells/tumors. Treatment of ZR-75 breast cancer cells with 17β-estradiol (E2) induced a greater than fourfold increase of VEGF mRNA levels. ZR-75 breast cancer cells were transfected with pVEGF1, a construct containing a −2018 to +50 VEGF promoter insert, and E2 induced reporter gene (luciferase) activity. Deletion and mutation analysis of the VEGF gene promoter identified a GC-rich region (−66 to −47) which was required for E2-induced transactivation of pVEGF5, a construct containing the minimal promoter (−66 to +54) that exhibited E2-responsiveness. Interactions of nuclear proteins from ZR-75 cells with the proximal GC-rich region of the VEGF gene promoter were investigated by electrophoretic mobility shift and chromatin immunoprecipitation assays. The results demonstrate that both Sp1 and Sp3 proteins bound the GC-rich motif (−66 to −47), and estrogen receptor α (ERα) interactions were confirmed by chromatin immunoprecipitation. Moreover, E2-dependent activation of constructs containing proximal and distal GC/GT-rich regions of the VEGF promoter was inhibited in ZR-75 cells transfected with small inhibitory RNAs for Sp1 and Sp3. These results were consistent with a mechanism of hormone activation of VEGF through ERα/Sp1 and ERα/Sp3 interactions with GC-rich motifs.

Small inhibitory RNA duplexes for Sp1 mRNA block basal and estrogen-induced gene expression and cell cycle progression in MCF-7 breast cancer cells

Small interfering RNA duplexes containing 21–22 nucleotides that mediate sequence-specific mRNA degradation and inhibitory RNA (iRNA) for Sp1 mRNA were used in this study to investigate the role of Sp1 on basal and hormone-induced growth and transactivation in MCF-7 and ZR-75 human breast cancer cells. Transfection of Sp1 iRNA in MCF-7 or ZR-75 cells for 36–44 h decreased Sp1 protein (50–70%) in nuclear extracts, and immunohistochemical analysis showed that the Sp1 protein in transfected MCF-7 cells was barely detectable. In cell cycle progression studies in MCF-7 cells, decreased Sp1 protein was accompanied by a decrease in cells in the S phase and an increase in cells in G0/G1, and estrogen-induced G0/G1 → S phase progression was inhibited in cells treated with iRNA for Sp1. Sp1 iRNA also specifically blocked basal and estrogen-induced transactivation in cells transfected with a GC-rich construct linked to a luciferase reporter gene (pSp13), and this was accompanied by decreased Sp1 binding to this GC-rich promoter as determined in gel mobility shift and chromatin immunoprecipitation assays. These results clearly demonstrate the key role of the Sp1 protein in basal and estrogen-induced growth and gene expression in breast cancer cells.

Oncogenic microRNA-27a is a target for anticancer agent methyl 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate in colon cancer cells

Methyl 2‐cyano‐3,11‐dioxo‐18β‐olean‐1,12‐dien‐30‐oate (CDODA‐Me) 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 G2/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 G2/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.

Synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid induces growth arrest in HER2-overexpressing breast cancer cells

HER2 overexpression is one of the most recognizable molecular alterations in breast tumors known to be associated with a poor prognosis. In the study described here, we explored the effect of HER2 overexpression on the sensitivity of breast cancer cells to the growth-inhibitory effects of 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), a synthetic triterpenoid, both in vitro and in vivo in a xenograft model of breast cancer. Both cell growth and colony formation in the soft agar assay, a hallmark of the transformation phenotype, were preferentially suppressed in HER2-overexpressing cell lines at low concentrations of CDDO, whereas growth-inhibitory effects at high concentrations did not correlate with the expression level of HER2. CDDO dose-dependently inhibited phosphorylation of HER2 in HER2-overexpressing cells and diminished HER2 kinase activity in vitro. CDDO induced the transactivation of the nuclear receptor peroxisome proliferator-activated receptor-γ in both vector control and HER2-transfected MCF7 cells. Dose-response studies showed that the growth inhibition seen at lower concentrations of CDDO correlated with induction of the tumor suppressor gene caveolin-1, which is known to inhibit breast cancer cell growth. CDDO also reduced cyclin D1 mRNA and protein expression. In vivo studies with liposomally encapsulated CDDO showed complete abrogation of the growth of the highly tumorigenic MCF7/HER2 cells in a xenograft model of breast cancer. These findings provide the first in vitro and in vivo evidence that CDDO effectively inhibits HER2 tyrosine kinase activity and potently suppresses the growth of HER2-overexpressing breast cancer cells and suggest that CDDO has a therapeutic potential in advanced breast cancer. [Mol Cancer Ther 2006;5(2):317–28]

Targeting Sp1 transcription factor in prostate cancer therapy

Transcription factors are proteins that regulate gene expression by binding to specific DNA sequences within gene promoter regions. Specificity protein (Sp) family transcription factors play a critical role in various cellular processes and have been shown to be associated with tumorigenesis. The Sp family consists of several members that contain a highly conserved DNA-binding domain composed of three zinc fingers at the C-terminus and serine/threonine- and glutamine-rich transactivation domains at the N-terminal. Sp1 is elevated in several cancers including prostate and is associated with the prognosis of patients. Sp1, Sp3, and Sp4 regulate a variety of cancer associated genes that are involved in cell cycle, proliferation, cell differentiation, and apoptosis. Studies have shown that in prostate cancer, Sp1 regulates important genes like androgen receptor, TGF-β, c-Met, fatty acid synthase, matrix metalloprotein (MT1-MMP), PSA, and α-integrin. These results highlight the importance of Sp1 in prostate cancer and emphasize the potential therapeutic value of targeting Sp1. Several strategies, including the use of natural and synthetic compounds, have been used to inhibit Sp1 in prostate cancer. These include polyphenol quercetin, betulinic acid, acetyl-11-keto-beta-boswellic acid, tea phenols, isothiocyanates, thiazolidinediones, arsenic trioxide, and selenium. This review will describe the association of Sp proteins in prostate cancer with a special emphasis on some of the agents tested to target Sp proteins for the treatment of this malignancy.

Methyl 2-cyano-3,12-dioxooleana-1,9-dien-28-oate decreases specificity protein transcription factors and inhibits pancreatic tumor growth: role of microRNA-27a.

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 Sp-regulated genes, growth inhibition, induction of apoptosis, and antiangiogenic responses.

Tolfenamic acid inhibits esophageal cancer through repression of specificity proteins and c-Met

The non-steroidal anti-inflammatory drug tolfenamic acid (TA) inhibits proliferation of SEG-1 and BIC-1 esophageal cancer cells with half-maximal growth inhibitory concentration values of 36 and 48 muM, respectively. TA also increased Annexin V staining in both cell lines, indicative of proapoptotic activity. Treatment of SEG-1 and BIC-1 cells with TA for up to 72 h decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and this was accompanied by decreased expression of the well-characterized Sp-regulated genes cyclin D1, vascular endothelial growth factor and survivin. TA also decreased hepatocyte growth factor receptor, (c-Met), a receptor tyrosine kinase that is overexpressed in esophageal cancer cells and tumors and is an important drug target. Knockdown of Sp1, Sp3 and Sp4 by RNA interference in SEG-1 and BIC-1 cells also decreased c-Met expression, demonstrating that c-Met is an Sp-regulated gene in esophageal cancer cells. Sp1 was overexpressed in esophageal cancer cells and tumors and increased Sp1 staining was observed in esophageal tumors from patients. TA (20 mg/kg/day) also decreased tumor growth and weight in athymic nude mice bearing SEG-1 cells as xenografts and this was accompanied by increased apoptosis and decreased Sp1 and c-Met staining in tumors from treated mice. Thus, TA-dependent downregulation of Sp transcription factors and c-Met defines a novel chemotherapeutic approach for treatment of esophageal cancer.

Regulation of KiSS-1 Metastasis Suppressor Gene Expression in Breast Cancer Cells by Direct Interaction of Transcription Factors Activator Protein-2α and Specificity Protein-1

KiSS-1 has been shown to function as a tumor metastasis suppressor gene and reduce the number of metastases in different cancers. The expression of KiSS-1 or KiSS1, like other tumor suppressor, is commonly reduced or completely ablated in a variety of cancers via an unknown mechanism. Here we show that the loss of KiSS-1 expression in highly metastatic breast cancer cell lines correlates directly with the expression levels of two transcription factors, activator protein-2α (AP-2α) and specificity protein 1 (Sp1), which synergistically activate the transcriptional regulation of KiSS-1 in breast cancer cells. Although the KiSS-1 promoter contains multiple AP-2α binding elements, AP-2α-mediated regulation occurs indirectly through Sp1 sites, as determined by deletion and mutation analysis. Overexpression of AP-2α into highly metastatic breast cell lines did not alter KiSS-1 promoter-driven luciferase gene activity. However, co-transfection of AP-2α wild-type or the dominant negative form of AP-2 lacking its C-terminal DNA-binding domain, AP-2B, together with Sp1, increased KiSS-1 promoter activity dramatically, suggesting that AP-2α regulation of KiSS-1 transcription does not require direct binding to the KiSS-1 promoter. Furthermore, we demonstrated that AP-2α directly interacted with Sp1 to form transcription complexes at two tandem Sp1-binding sites of the promoter to activate KiSS-1 transcription. Together, our results indicate that AP-2α and Sp1 are strong transcriptional regulators of KiSS-1 and that loss or decreased expression of AP-2α in breast cancer may account for the loss of tumor metastasis suppressor KiSS-1 expression and thus increased cancer metastasis.