Shengxi Liu

Aryl hydrocarbon receptor agonists directly activate estrogen receptor α in MCF-7 breast cancer cells

Abstract The aryl hydrocarbon receptor (AhR) binds with high affinity to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatics, but also binds with lower affinity to structurally diverse exogenous and endogenous chemicals. One study reported that 3-methylcholanthrene (3MC) activated the estrogen receptor (ER) through the AhR, which acts as co-regulatory protein, whereas a recent report showed that 3MC directly bound and activated ERα. This study also shows that the AhR agonists benzo[a]pyrene, 3,3′,4,4′-tetrachlorobiphenyl, chrysin, 6-methyl-1,3,8-trichlorodibenzofuran, and 3,3′-diindolylmethane also induce ERα-dependent transactivation. Moreover, in chromatin immunoprecipitation assays, these compounds induce binding of AhR and ERα to the CYP1A1 and pS2 gene promoters, which is consistent with their activities as both selective AhR modulators (SAhRMs) and selective ER modulators (SERMs).

Induction of endoplasmic reticulum-induced stress genes in Panc-1 pancreatic cancer cells is dependent on Sp proteins.

Endoplasmic reticulum (ER) stress plays a critical role in multiple diseases, and pharmacologically active drugs can induce cell death through ER stress pathways. Stress-induced genes are activated through assembly of transcription factors on ER stress response elements (ERSEs) in target gene promoters. Gel mobility shift and chromatin immunoprecipitation assays have confirmed interactions of NF-Y and YY1 with the distal motifs of the tripartite ERSE from the glucose-related protein 78 (GRP78) gene promoter. The GC-rich nonanucleotide (N9) sequence, which forms the ER stress response binding factor (ERSF) complex binds TFII-I and ATF6; however, we have now shown that in Panc-1 pancreatic cancer cells, this complex also binds Sp1, Sp3, and Sp4 proteins. Sp proteins are constitutively bound to the ERSE; however, activation of GRP78 protein (or reporter gene) by thapsigargin or tunicamycin is inhibited after cotransfection with small inhibitory RNAs for Sp1, Sp3, and Sp4. This study demonstrates that Sp transcription factors are important for stress-induced responses through their binding to ERSEs.

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]

Role of specificity protein transcription factors in estrogen-induced gene expression in MCF-7 breast cancer cells

Deletion analysis of several 17beta-estradiol (E(2))-responsive genes have identified GC-rich sites that are associated with hormone-induced transactivation in MCF-7 breast cancer cells. However, the role of individual specificity proteins (Sps) in mediating hormone-induced gene expression has not been unequivocally determined. In transient transfection studies using E(2)-responsive GC-rich promoters from the E(2)F1, carbamoylphosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD), and retinoic acid receptor alpha (RAR alpha) genes, RNA interference using small inhibitory RNAs for Sp1 (iSp1), Sp3 (iSp3), and Sp4 (iSp4) decreased both basal and E(2)-induced transactivation. The contributions of individual Sp proteins to basal and E(2)-induced activity were promoter dependent. iSp1, iSp3, and iSp4 also significantly inhibited hormonal induction of E(2)F1, CAD, and RAR alpha mRNA levels; however, the enhanced inhibitory effects of the latter two small inhibitory RNAs suggest that Sp3 and Sp4 play a major role in estrogen receptor alpha/Sp-mediated gene expression in MCF-7 cells.

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.

17β-estradiol (E2) induces cdc25A gene expression in breast cancer cells by genomic and non-genomic pathways

Cdc25A is a potent tyrosine phosphatase that catalyzes specific dephosphorylation of cyclin/cyclin‐dependent kinase (cdk) complexes to regulate G1 to S‐phase cell cycle progression. Cdc25A mRNA levels are induced by 17β‐estradiol (E2) in ZR‐75 breast cancer cells, and deletion analysis of the cdc25A promoter identified the −151 to −12 region as the minimal E2‐responsive sequence. Subsequent mutation/deletion analysis showed that at least three different cis‐elements were involved in activation of cdc25A by E2, namely, GC‐rich Sp1 binding sites, CCAAT motifs that bind NF‐Y, and E2F sites that bind DP/E2F1 proteins. Studies with inhibitors and dominant negative expression plasmids show that E2 activates cdc25A expression through activation of genomic ERα/Sp1 and E2F1 and cAMP‐dependent activation of NF‐YA. Thus, both genomic and non‐genomic pathways of estrogen action are involved in induction of cdc25A in breast cancer cells. J. Cell. Biochem.

5,5′-Dibromo-bis(3′-indolyl)methane induces Krüppel-like factor 4 and p21 in colon cancer cells

Bis(3′-indolyl)methane (DIM) is a metabolite of the phytochemical indole-3-carbinol, and both compounds exhibit a broad spectrum of anticancer activities. We have developed a series of synthetic symmetrical ring-substituted DIM analogues, including 5,5′-dibromoDIM, which are more potent than DIM as inhibitors of cancer cell and tumor growth. In colon cancer cells, 5,5′-dibromoDIM decreased cell proliferation and inhibited G0-G1- to S-phase progression, and this was accompanied by induction of the cyclin-dependent kinase inhibitor p21 in HT-29 and RKO colon cancer cells. Mechanistic studies showed that induction of p21 in both RKO (p53 wild-type) and HT-29 (p53 mutant) cells by 5,5′-dibromoDIM was Krüppel-like factor 4 (KLF4) dependent, and induction of p53 in RKO cells was also KLF4 dependent. Analysis of the p21 promoter in p53-dependent RKO cells showed that 5,5′-dibromoDIM activated p21 gene expression through the proximal GC-rich sites 1 and 2, and chromatin immunoprecipitation assays showed that KLF4 and p53 bound to this region of the promoter, whereas in HT-29 cells unidentified upstream cis-elements were required for induction of p21. 5,5′-DibromoDIM (30 mg/kg/d) also inhibited tumor growth and induced p21 in athymic nude mice bearing RKO cells as xenografts, showing that ring-substituted DIM such as 5,5′-dibromoDIM represent a novel class of mechanism-based drugs for clinical treatment of colon cancer. [Mol Cancer Ther 2008;7(7):2109–20]