Kyungsil Yoon

Activation of nerve growth factor-induced Bα by methylene-substituted diindolylmethanes in bladder cancer cells induces apoptosis and inhibits tumor growth

Nerve growth factor-induced B (NGFI-B) genes are orphan nuclear receptors, and NGFI-Bα (Nur77, TR3) is overexpressed in bladder tumors and bladder cancer cells compared with nontumorous bladder tissue. 1,1-Bis(3′-indolyl)-1-(p-methoxyphenyl)-methane (DIM-C-pPhOCH3) and 1,1-bis(3′-indolyl)-1-(p-phenyl)methane have previously been identified as activators of Nur77, and both compounds inhibited growth and induced apoptosis of UC-5 and KU7 bladder cancer cells. The proapoptotic effects of methylene-substituted diindolylmethanes (C-DIMs) were unaffected by cotreatment with leptomycin B and were dependent on nuclear Nur77, and RNA interference with a small inhibitory RNA for Nur77 (iNur77) demonstrated that C-DIM-induced activation of apoptosis was Nur77-dependent. Microarray analysis of DIM-C-pPhOCH3-induced genes in UC-5 bladder cancer cells showed that this compound induced multiple Nur77-dependent proapoptotic or growth inhibitory genes including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), cystathionase, p21, p8, and sestrin-2. DIM-C-pPhOCH3 (25 mg/kg/d) also induced apoptosis and inhibited tumor growth in athymic nude mice bearing KU7 cells as xenografts, demonstrating that Nur77-active C-DIMs exhibit potential for bladder cancer chemotherapy by targeting Nur77, which is overexpressed in this tumor type.

Genetic Ablation of CCAAT/Enhancer Binding Protein α in Epidermis Reveals Its Role in Suppression of Epithelial Tumorigenesis

CCAAT/enhancer binding protein alpha (C/EBPalpha) is a basic leucine zipper transcription factor that inhibits cell cycle progression and regulates differentiation in various cell types. C/EBPalpha is inactivated by mutation in acute myeloid leukemia (AML) and is considered a human tumor suppressor in AML. Although C/EBPalpha mutations have not been observed in malignancies other than AML, greatly diminished expression of C/EBPalpha occurs in numerous human epithelial cancers including lung, liver, endometrial, skin, and breast, suggesting a possible tumor suppressor function. However, direct evidence for C/EBPalpha as an epithelial tumor suppressor is lacking due to the absence of C/EBPalpha mutations in epithelial tumors and the lethal effect of C/EBPalpha deletion in mouse model systems. To examine the function of C/EBPalpha in epithelial tumor development, an epidermal-specific C/EBPalpha knockout mouse was generated. The epidermal-specific C/EBPalpha knockout mice survived and displayed no detectable abnormalities in epidermal keratinocyte proliferation, differentiation, or apoptosis, showing that C/EBPalpha is dispensable for normal epidermal homeostasis. In spite of this, the epidermal-specific C/EBPalpha knockout mice were highly susceptible to skin tumor development involving oncogenic Ras. These mice displayed decreased tumor latency and striking increases in tumor incidence, multiplicity, growth rate, and the rate of malignant progression. Mice hemizygous for C/EBPalpha displayed an intermediate-enhanced tumor phenotype. Our results suggest that decreased expression of C/EBPalpha contributes to deregulation of tumor cell proliferation. C/EBPalpha had been proposed to block cell cycle progression through inhibition of E2F activity. We observed that C/EBPalpha blocked Ras-induced and epidermal growth factor-induced E2F activity in keratinocytes and also blocked Ras-induced cell transformation and cell cycle progression. Our study shows that C/EBPalpha is dispensable for epidermal homeostasis and provides genetic evidence that C/EBPalpha is a suppressor of epithelial tumorigenesis.

p21 Expression Is Induced by Activation of Nuclear Nerve Growth Factor–Induced Bα (Nur77) in Pancreatic Cancer Cells

1,1-Bis(3′-indolyl)-1-(p-anisyl)methane (DIM-C-pPhOCH3) activates the orphan receptor nerve growth factor–induced Bα (Nur77) in cancer cells, and in this study, DIM-C-pPhOCH3 decreased Panc1 pancreatic cancer cell survival and arrested cells in G0-G1. These responses were accompanied by induction of the cyclin-dependent kinase inhibitor p21 in pancreatic cancer cells. Mechanistic studies showed that induction of p21 mRNA and protein by DIM-C-pPhOCH3 was Nur77 dependent but did not depend on Krüppel-like factor 4, which was also induced by DIM-C-pPhOCH3. Activation of p21 promoter constructs by DIM-C-pPhOCH3 required the GC-rich proximal region of the promoter, and results of RNA interference studies showed that Nur77-dependent activation of the p21 promoter involved interactions with Sp1 and Sp4 but not Sp3. Interactions of Nur77 with the p21 promoter in Panc1 cells treated with DIM-C-pPhOCH3 were also confirmed in chromatin immunoprecipitation assays. These data show that activation of nuclear Nur77 results in a novel pathway for induction of p21, which is independent of Nur77 response elements but dependent on Sp proteins bound to the GC-rich proximal region of the p21 promoter. [Mol Cancer Res 2009;7(7):1169–78]

1,1‐bis(3′‐indolyl)‐1‐(p‐methoxyphenyl)methane activates Nur77‐independent proapoptotic responses in colon cancer cells

1,1‐Bis(3′‐indolyl)‐1‐(p‐methoxyphenyl)methane (DIM‐C‐pPhOCH3) is a methylene‐substituted diindolylmethane (C‐DIM) analog that activates the orphan receptor nerve growth factor‐induced‐Bα (NGFI‐Bα, Nur77). RNA interference studies with small inhibitory RNA for Nur77 demonstrate that DIM‐C‐pPhOCH3 induces Nur77‐dependent and ‐independent apoptosis, and this study has focused on delineating the Nur77‐independent proapoptotic pathways induced by the C‐DIM analog. DIM‐C‐pPhOCH3 induced caspase‐dependent apoptosis in RKO colon cancer cells through decreased mitochondrial membrane potential which is accompanied by increased mitochondrial bax/bcl‐2 ratios and release of cytochrome c into the cytosol. DIM‐C‐pPhOCH3 also induced phosphatidylinositol‐3‐kinase‐dependent activation of early growth response gene‐1 which, in turn, induced expression of the proapoptotic nonsteroidal anti‐inflammatory drug‐activated gene‐1 (NAG1) in RKO and SW480 colon cancer cells. Moreover, DIM‐C‐pPhOCH3 also induced NAG‐1 expression in colon tumors in athymic nude mice bearing RKO cells as xenografts. DIM‐C‐pPhOCH3 also activated the extrinsic apoptosis pathway through increased phosphorylation of c‐jun N‐terminal kinase which, in turn, activated C/EBP homologous transcription factor (CHOP) and death receptor 5 (DR5). Thus, the effectiveness of DIM‐C‐pPhOCH3 as a tumor growth inhibitor is through activation of Nur77‐dependent and ‐independent pathways.