The Role of Endocrine Disruption Chemical-Regulated Aryl Hydrocarbon Receptor Activity in the Pathogenesis of Pancreatic Diseases and Cancer.

The aryl hydrocarbon receptor (AHR) serves as a ligand-activated transcription factor crucial for regulating fundamental cellular and molecular processes, such as xenobiotic metabolism, immune responses, and cancer development. Notably, a spectrum of endocrine-disrupting chemicals (EDCs) act as agonists or antagonists of AHR, leading to the dysregulation of pivotal cellular and molecular processes and endocrine system disruption. Accumulating evidence suggests a correlation between EDC exposure and the onset of diverse pancreatic diseases, including diabetes, pancreatitis, and pancreatic cancer. Despite this association, the mechanistic role of AHR as a linchpin molecule in EDC exposure-related pathogenesis of pancreatic diseases and cancer remains unexplored. This review comprehensively examines the involvement of AHR in EDC exposure-mediated regulation of pancreatic pathogenesis, emphasizing AHR as a potential therapeutic target for the pathogenesis of pancreatic diseases and cancer.

Regulation of a long noncoding RNA MALAT1 by aryl hydrocarbon receptor in pancreatic cancer cells and tissues.

Environmental toxicants such as dioxins and polycyclic aromatic carbons are risk factors for pancreatitis and pancreatic cancer. These toxicants activate aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, of which activation regulates many downstream biological events, including xenobiotic metabolism, inflammation, and cancer cell growth and transformation. Here, we identified that environmental toxicant-activated AHR increased expression of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in pancreatic cancer cells and pancreatic tissues. The MALAT1 is a long noncoding (lnc) RNA which interacts with Enhancer of Zeste 2 (EZH2), a histone methyltransferase with epigenetic silencer activity, and the MALAT1-EZH2 interaction increased its epigenetic silencing activity. In contrast, AHR antagonist, CH223191 or resveratrol, counteracted the AHR-mediated MALAT1 induction and MALAT1-enahnced EZH2 activity. Collectively, these results revealed a novel pathway of how environmental exposure leads to epigenetic alteration via activation of AHR-MALAT1-EZH2 signaling axis under pancreatic tissue- and cancer cell-context.

NR2E3 is a key component in p53 activation by regulating a long noncoding RNA DINO in acute liver injuries.

Damage-induced long noncoding RNA (DINO) is a long noncoding RNA that directly interacts with p53 and thereby enhances p53 stability and activity in response to various cellular stresses. Here, we demonstrate that nuclear receptor subfamily 2 group E member 3 (NR2E3) plays a crucial role in maintaining active DINO epigenetic status for its proper induction and subsequent p53 activation. In acetaminophen (APAP)- or carbon tetrachloride-induced acute liver injuries, NR2E3 knockout (KO) mice exhibited far more severe liver injuries due to impaired DINO induction and p53 activation. Mechanistically, NR2E3 loss both in vivo and in vitro induced epigenetic DINO repression accompanied by reduced DINO chromatin accessibility. Furthermore, compared with the efficient reversal by a typical antidote N-acetylcysteine (NAC) treatment of APAP-induced liver injury in wild-type mice, the liver injury of NR2E3 KO mice was not effectively reversed, indicating that an intact NR2E3-DINO-p53-signaling axis is essential for NAC-mediated recovery against APAP-induced hepatotoxicity. These findings establish that NR2E3 is a critical component in p53 activation and a novel susceptibility factor to drug- or toxicant-induced acute liver injuries.-Khanal, T., Leung, Y.-K., Jiang, W., Timchenko, N., Ho, S.-M., Kim, K. NR2E3 is a key component in p53 activation by regulating a long noncoding RNA DINO in acute liver injuries.

The Loss of an Orphan Nuclear Receptor NR2E3 Augments Wnt/ß-catenin Signaling via Epigenetic Dysregulation that Enhances Sp1-ß catenin-p300 Interactions in Hepatocellular Carcinoma.

The orphan nuclear receptor NR2E3 (Nuclear receptor subfamily 2 group E, Member 3) is an epigenetic player that modulates chromatin accessibility to activate p53 during liver injury. Nonetheless, a precise tumor suppressive and epigenetic role of NR2E3 in hepatocellular carcinoma (HCC) development remains unclear. HCC patients expressing low NR2E3 exhibit unfavorable clinical outcomes, aligning with heightened activation of the Wnt/ß-catenin signaling pathway. The murine HCC models utilizing NR2E3 knockout mice consistently exhibits accelerated liver tumor formation accompanied by enhanced activation of Wnt/ß-catenin signaling pathway and inactivation of p53 signaling. At cellular level, the loss of NR2E3 increases the acquisition of aggressive cancer cell phenotype and tumorigenicity and upregulates key genes in the WNT/ß-catenin pathway with increased chromatin accessibility. This event is mediated through increased formation of active transcription complex involving Sp1, ß-catenin, and p300, a histone acetyltransferase, on the promoters of target genes. These findings demonstrate that the loss of NR2E3 activates Wnt/ß-catenin signaling at cellular and organism levels and this dysregulation is associated with aggressive HCC development and poor clinical outcomes. In summary, NR2E3 is a novel tumor suppressor with a significant prognostic value, maintaining epigenetic homeostasis to suppress the Wnt/ß-catenin signaling pathway that promotes HCC development.

Inhibition of rhabdomyosarcoma cell and tumor growth by targeting specificity protein (Sp) transcription factors.

Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are highly expressed in rhabdomyosarcoma (RMS) cells. In tissue arrays of RMS tumor cores from 71 patients, 80% of RMS patients expressed high levels of Sp1 protein, whereas low expression of Sp1 was detected in normal muscle tissue. The non-steroidal anti-inflammatory drug (NSAID) tolfenamic acid (TA) inhibited growth and migration of RD and RH30 RMS cell lines and also inhibited tumor growth in vivo using a mouse xenograft (RH30 cells) model. The effects of TA were accompanied by downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes in RMS cells and tumors, and the role of Sp protein downregulation in mediating inhibition of RD and RH30 cell growth and migration was confirmed by individual and combined knockdown of Sp1, Sp3 and Sp4 proteins by RNA interference. TA treatment and Sp knockdown in RD and RH30 cells also showed that four genes that are emerging as individual drug targets for treating RMS, namely c-MET, insulin-like growth factor receptor (IGFR), PDGFRα and CXCR4, are also Sp-regulated genes. These results suggest that NSAIDs such as TA may have potential clinical efficacy in drug combinations for treating RMS patients.

FOXM1 mediates Dox resistance in breast cancer by enhancing DNA repair.

Transcription factors are direct effectors of altered signaling pathways in cancer and frequently determine clinical outcomes in cancer patients. To uncover new transcription factors that would determine clinical outcomes in breast cancer, we systematically analyzed gene expression data from breast cancer patients. Our results revealed that Forkhead box protein M1 (FOXM1) is the top-ranked survival-associated transcription factor in patients with triple-negative breast cancer. Surprisingly, silencing FOXM1 expression led breast cancer cells to become more sensitive to doxorubicin (Dox). We found that FOXM1-dependent resistance to Dox is mediated by regulating DNA repair genes. We further demonstrated that NFκB1 interacts with FOXM1 in the presence of Dox to protect breast cancer cells from DNA damage. Finally, silencing FOXM1 expression in breast cancer cells in a mouse xenograft model significantly sensitized the cells to Dox. Our systematic approaches identified an unexpected role of FOXM1 in Dox resistance by regulating DNA repair genes, and our findings provide mechanistic insights into how FOXM1 mediates resistance to Dox and evidence that FOXM1 may be a promising therapeutic target for sensitizing breast cancer cells to Dox.

Curcumin and synthetic analogs induce reactive oxygen species and decreases specificity protein (Sp) transcription factors by targeting microRNAs.

BACKGROUND: Curcumin inhibits growth of several cancer cell lines, and studies in this laboratory in bladder and pancreatic cancer cells show that curcumin downregulates specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and pro-oncogenic Sp-regulated genes. In this study, we investigated the anticancer activity of curcumin and several synthetic cyclohexanone and piperidine analogs in colon cancer cells. METHODS: The effects of curcumin and synthetic analogs on colon cancer cell proliferation and apoptosis were determined using standardized assays. The changes in Sp proteins and Sp-regulated gene products were analysed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a), miR-20a, miR-17-5p and ZBTB10 and ZBTB4 mRNA expression. RESULTS: The IC50 (half-maximal) values for growth inhibition (24 hr) of colon cancer cells by curcumin and synthetic cyclohexanone and piperidine analogs of curcumin varied from 10 µM for curcumin to 0.7 µM for the most active synthetic piperidine analog RL197, which was used along with curcumin as model agents in this study. Curcumin and RL197 inhibited RKO and SW480 colon cancer cell growth and induced apoptosis, and this was accompanied by downregulation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and Sp-regulated genes including the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET), survivin, bcl-2, cyclin D1 and NFκB (p65 and p50). Curcumin and RL197 also induced reactive oxygen species (ROS), and cotreatment with the antioxidant glutathione significantly attenuated curcumin- and RL197-induced growth inhibition and downregulation of Sp1, Sp3, Sp4 and Sp-regulated genes. The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR)-27a, miR-20a and miR-17-5p that regulate these repressors. CONCLUSIONS: These results identify a new and highly potent curcumin derivative and demonstrate that in cells where curcumin and RL197 induce ROS, an important underlying mechanism of action involves perturbation of miR-ZBTB10/ZBTB4, resulting in the induction of these repressors which downregulate Sp transcription factors and Sp-regulated genes.

The androgen receptor inhibits transcription of GPER1 by preventing Sp1 and Sp3 from binding to the promoters in prostate cancer cells.

G-1, a GPER1 agonist, was shown to inhibit the growth of castration-resistant mouse xenografts but not their parental androgen-dependent tumors. It is currently unknown how the androgen receptor (AR) represses GPER1 expression. Here, we found that two GPER1 mRNA variants (GPER1v2 and GPER1v4) were transcriptionally repressed, not via transcript destabilization, by the androgen-activated AR. Although no AR binding was found in all active promoters near GPER1, data from promoter assays suggested that both variants' promoters were inhibited by androgen treatment. Site-directed mutagenesis on Sp1/Sp3 binding sites revealed their role in supporting the basal expression of GPER1. Knockdown of Sp1 and Sp3 together but not separately repressed GPER1 expression whereas overexpression of both Sp1 and Sp3 together was required to alleviate AR repression of GPER1. Based on the chromatin immunoprecipitation data, Sp3 was found to bind to the promoters prior to the binding of Sp1 and RNA polymerase II. However, the binding of all three transcription factors was inhibited by DHT treatment. Concordantly, DHT treatment induced nuclear interactions between AR and Sp1 or Sp3. Taken together, these results indicate that AR represses transcription of GPER1 by binding to Sp1 and Sp3 independently to prevent their transactivation of the GPER1 promoters.

Activation of nuclear TR3 (NR4A1) by a diindolylmethane analog induces apoptosis and proapoptotic genes in pancreatic cancer cells and tumors.

NR4A1 (Nur77, TR3) is overexpressed in pancreatic tumors and activation of TR3 by 1,1-bis(3'-indolyl)-1-(p-methoxyphenyl)methane (DIM-C-pPhOCH(3)) inhibits cell and tumor growth and induces apoptosis. Microarray analysis demonstrates that in L3.6pL pancreatic cancer cells DIM-C-pPhOCH(3) induces genes associated with metabolism, homeostasis, signal transduction, transcription, stress, transport, immune responses, growth inhibition and apoptosis. Among the most highly induced growth inhibitory and proapoptotic genes including activating transcription factor 3 (ATF3), p21, cystathionase, dual specificity phosphatase 1 and growth differentiation factor 15, RNA interference studies demonstrated that induction of all but the later gene by DIM-C-pPhOCH(3) were TR3-dependent. We also observed that DIM-C-pPhOCH(3) induced Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and induction of TRAIL was ATF3 dependent. Results of this and previous studies demonstrate that TR3 is unique among nuclear receptors since nuclear TR3 is activated or deactivated by diindolylmethane derivatives to induce different apoptotic and growth inhibitory pathways that inhibit pancreatic cancer cell and tumor growth.

In vivo profiling of estrogen receptor/specificity protein-dependent transactivation.

17beta-Estradiol (E2) activates the estrogen receptor (ER) through multiple genomic and nongenomic pathways in various tissues/organs. ERalpha/specificity protein-dependent activation of E2-responsive genes containing GC-rich promoters has been identified in breast and other cancer cell lines, and in this study, we describe transgenic animals overexpressing a transgene containing three tandem GC-rich sites linked to a minimal TATA or thymidine kinase promoter and a luciferase gene. Several mouse lines expressing the transgenes were characterized and, in line 15, E2 induced a 9-fold increase in luciferase activity in the female mouse uterus, and the synthetic estrogens bisphenol A and nonylphenol also induced uterine luciferase activity. The pure antiestrogen ICI 182,780 induced luciferase activity in the mouse uterus, and similar results were observed for ICI 182,780 in breast cancer cells transfected with this construct. Differences in the ER agonist and antagonist activities of E2, nonylphenol, bisphenol A, and ICI 182,780 were investigated in the male testis and penis and the male and female stomach in line 15 transgenic mice. All of these tissues were hormone responsive; however, the patterns of induced or repressed luciferase activity were ligand structure, tissue, and sex dependent. These results demonstrate for the first time hormonal activation or repression of a GC-rich promoter in vivo, and the results suggest that the ERalpha/specificity protein pathway may contribute to E2-dependent induction and repression of genes.

3-Methylcholanthrene and other aryl hydrocarbon receptor agonists directly activate estrogen receptor alpha.

3-Methylcholanthrene (3MC) is an aryl hydrocarbon receptor (AhR) agonist, and it has been reported that 3MC induces estrogenic activity through AhR-estrogen receptor alpha (ER alpha) interactions. In this study, we used 3MC and 3,3',4,4',5-pentachlorobiphenyl (PCB) as prototypical AhR ligands, and both compounds activated estrogen-responsive reporter genes/gene products (cathepsin D) in MCF-7 breast cancer cells. The estrogenic responses induced by these AhR ligands were inhibited by the antiestrogen ICI 182780 and by the transfection of a small inhibitory RNA for ER alpha but were not affected by the small inhibitory RNA for AhR. These results suggest that 3MC and PCB directly activate ER alpha, and this was confirmed in a competitive ER alpha binding assay and in a fluorescence resonance energy transfer experiment in which PCB and 3MC induced CFP-ER alpha/YFP-ER alpha interactions. In a chromatin immunoprecipitation assay, PCB and 3MC enhanced ER alpha (but not AhR) association with the estrogen-responsive region of the pS2 gene promoter. Moreover, in AhR knockout mice, 3MC increased uterine weights and induced expression of cyclin D1 mRNA levels. These results show that PCB and 3MC directly activate ER alpha-dependent transactivation and extend the number of ligands that activate both AhR and ER alpha.

Molecular mechanism of inhibitory aryl hydrocarbon receptor-estrogen receptor/Sp1 cross talk in breast cancer cells.

The trifunctional carbamoylphosphate synthetase/aspartate transcarbamyltransferase/dihydroorotase (CAD) gene is hormone responsive in MCF-7 and ZR-75 breast cancer cells, and this response is inhibited by the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Estrogen-dependent induction of CAD mRNA and reporter gene activity in cells transfected with constructs (pCAD) containing hormone-responsive GC-rich CAD promoter inserts involves estrogen receptor alpha (ERalpha)/Sp1 interactions with these proximal GC-rich motifs. TCDD also inhibits hormone-induced transactivation in MCF-7 and ZR-75 cells transfected with pCAD constructs. The mechanism of inhibitory AhR-ERalpha/Sp1 cross talk was further investigated by chromatin immunoprecipitation (ChIP), and the results show that ERalpha/Sp1 and the AhR are constitutively bound to the CAD gene promoter and only minor changes are observed after treatment with 17beta-estradiol, TCDD, or their combination. However, examination of interactions of these transcription factors by fluorescence resonance energy transfer shows that E2 enhances ERalpha-Sp1 interactions, whereas cotreatment with TCDD significantly decreases interaction of these proteins. These results suggest that inhibitory AhR-ERalpha/Sp1 cross talk is due, in part, to enhanced association of AhR and ERalpha (also determined by fluorescence resonance energy transfer), which coordinately dissociates ER and Sp1 and decreases ERalpha/Sp1-mediated transactivation, whereas remaining associated with the CAD promoter. This represents a novel interaction between two ligand activated receptors where one receptor inhibits activation of the second receptor.

Loss of NR2E3 represses AHR by LSD1 reprogramming, is associated with poor prognosis in liver cancer.

The aryl hydrocarbon receptor (AHR) plays crucial roles in inflammation, metabolic disorder, and cancer. However, the molecular mechanisms regulating AHR expression remain unknown. Here, we found that an orphan nuclear NR2E3 maintains AHR expression, and forms an active transcriptional complex with transcription factor Sp1 and coactivator GRIP1 in MCF-7 human breast and HepG2 liver cancer cell lines. NR2E3 loss promotes the recruitment of LSD1, a histone demethylase of histone 3 lysine 4 di-methylation (H3K4me2), to the AHR gene promoter region, resulting in repression of AHR expression. AHR expression and responsiveness along with H3K4me2 were significantly reduced in the livers of Nr2e3rd7 (Rd7) mice that express low NR2E3 relative to the livers of wild-type mice. SP2509, an LSD1 inhibitor, fully restored AHR expression and H3K4me2 levels in Rd7 mice. Lastly, we demonstrated that both AHR and NR2E3 are significantly associated with good clinical outcomes in liver cancer. Together, our results reveal a novel link between NR2E3, AHR, and liver cancer via LSD1-mediated H3K4me2 histone modification in liver cancer development.

Analysis of estrogen receptor alpha-Sp1 interactions in breast cancer cells by fluorescence resonance energy transfer.

Estrogen-dependent regulation of several genes associated with cell cycle progression, proliferation, and nucleotide metabolism in breast cancer cells is associated with interactions of estrogen receptor (ER)alpha/Sp1 with GC-rich promoter elements. This study investigates ligand-dependent interactions of ERalpha and Sp1 in MCF-7 breast cancer cells using fluorescence resonance energy transfer (FRET). Chimeric ERalpha and Sp1 proteins fused to cyan fluorescent protein or yellow fluorescent protein were transfected into MCF-7 cells, and a FRET signal was induced after treatment with 17beta-estradiol, 4'-hydroxytamoxifen, or ICI 182,780. Induction of FRET by these ERalpha agonists/antagonists was paralleled by their activation of gene expression in cells transfected with a construct (pSp1(3)) containing three tandem Sp1 binding sites linked to a luciferase reporter gene. In contrast, interactions between ERalpha and Sp1DeltaDBD [a DNA binding domain (DBD) deletion mutant of Sp1] are not observed, and this is consistent with the critical role of the C-terminal DBD of Sp1 for interaction with ERalpha. Results of the FRET assay are consistent with in vitro studies on ERalpha/Sp1 interactions and transactivation, and confirm that ERalpha and Sp1 interact in living breast cancer cells.

Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are non-oncogene addiction genes in cancer cells.

Specificity protein (Sp) transcription factor (TF) Sp1 is overexpressed in multiple tumors and is a negative prognostic factor for patient survival. Sp1 and also Sp3 and Sp4 are highly expressed in cancer cells and in this study, we have used results of RNA interference (RNAi) to show that the three TFs individually play a role in the growth, survival and migration/invasion of breast, kidney, pancreatic, lung and colon cancer cell lines. Moreover, tumor growth in athymic nude mice bearing L3.6pL pancreatic cancer cells as xenografts were significantly decreased in cells depleted for Sp1, Sp3 and Sp4 (combined) or Sp1 alone. Ingenuity Pathway Analysis (IPA) of changes in gene expression in Panc1 pancreatic cancer cells after individual knockdown of Sp1, Sp3 and Sp4 demonstrates that these TFs regulate genes and pathways that correlated with the functional responses observed after knockdown but also some genes and pathways that inversely correlated with the functional responses. However, causal IPA analysis which integrates all pathway-dependent changes in all genes strongly predicted that Sp1-, Sp3- and Sp4-regulated genes were associated with the pro-oncogenic activity. These functional and genomic results coupled with overexpression of Sp transcription factors in tumor vs. non-tumor tissues and decreased Sp1 expression with age indicate that Sp1, Sp3 and Sp4 are non-oncogene addiction (NOA) genes and are attractive drug targets for individual and combined cancer chemotherapies.

Domains of estrogen receptor alpha (ERalpha) required for ERalpha/Sp1-mediated activation of GC-rich promoters by estrogens and antiestrogens in breast cancer cells.

Estrogen receptor alpha (ERalpha)/Sp1 activation of GC-rich gene promoters in breast cancer cells is dependent, in part, on activation function 1 (AF1) of ERalpha, and this study investigates contributions of the DNA binding domain (C) and AF2 (DEF) regions of ERalpha on activation of ERalpha/Sp1. 17Beta-estradiol (E2) and the antiestrogens 4-hydroxytamoxifen and ICI 182,780 induced reporter gene activity in MCF-7 and MDA-MB-231 cells cotransfected with human or mouse ERalpha (hERalpha or MOR), but not ERbeta and GC-rich constructs containing three tandem Sp1 binding sites (pSp13) or other E2-responsive GC-rich promoters. Estrogen and antiestrogen activation of hERalpha/Sp1 was dependent on overlapping and different regions of the C, D, E, and F domains of ERalpha. Antiestrogen-induced activation of hERalpha/Sp1 was lost using hERalpha mutants deleted in zinc finger 1 [amino acids (aa) 185-205], zinc finger 2 (aa 218-245), and the hinge/helix 1 (aa 265-330) domains. In contrast with antiestrogens, E2-dependent activation of hERalpha/Sp1 required the C-terminal F domain (aa 579-595), which contains a beta-strand structural motif. Moreover, in peptide competition experiments overexpression of a C-terminal (aa 575-595) F domain peptide specifically blocked E2-dependent activation of hERalpha/Sp1, suggesting that F domain interactions with nuclear cofactors are required for ERalpha/Sp1 action.

Deregulation of NR2E3, an orphan nuclear receptor, by benzo(a)pyrene-induced oxidative stress is associated with histone modification status change of the estrogen receptor gene promoter.

We previously reported that NR2E3, an orphan nuclear receptor, plays an important role in maintaining the basal expression of estrogen receptor α (ER) and that the NR2E3 level is highly correlated with the relapse-free survival of breast cancer patients. Here, we investigated the role of NR2E3 in benzo(a)pyrene (BaP)-mediated cell injury. BaP treatment reduced NR2E3 homo-dimer formation and expression and subsequently decreased ER expression. The chromatin immunoprecipitation assay results showed that the treatment of MCF-7 breast cancer cells and the mouse liver with BaP released NR2E3 from the ER promoter to transform the transcriptionally active histone modification status into a repressive state. NR2E3 depletion in MCF-7 cells also induced a similar inactive epigenetic status in the ER promoter region, indicating that NR2E3 is an essential epigenetic player that maintains basal ER expression. Interestingly, these negative effects of BaP on the expression levels of NR2E3 and ER were rescued by antioxidant treatment. Collectively, our study provides novel evidence to show that BaP-induced oxidative stress decreases ER expression, in part by regulating NR2E3 function, which modulates the epigenetic status of the ER promoter. NR2E3 is likely an essential epigenetic player that maintains basal ER expression to protect cells from BaP-induced oxidative injury.

Specificity protein (Sp) transcription factors and metformin regulate expression of the long non-coding RNA HULC.

Specificity protein 1 (Sp1) transcription factor (TF) regulates expression of long non-coding RNAs (lncRNAs) in hepatocellular carcinoma (HCC) cells. RNA interference (RNAi) studies showed that among several lncRNAs expressed in HepG2, SNU-449 and SK-Hep-1 cells, highly upregulated in liver cancer (HULC) was regulated not only by Sp1 but also Sp3 and Sp4 in the three cell lines. Knockdown of Sp transcription factors and HULC by RNAi showed that they play important roles in HCC cell proliferation, survival and migration. The relative contribution of Sp1, Sp3, Sp4 and HULC on these responses in HepG2, SNU-449 and SK-Hep-1 cells were cell context- and response-dependent. In the poorly differentiated SK-Hep-1 cells, knockdown of Sp1 or HULC resulted in genomic and morphological changes, indicating that Sp1 and Sp1-regulated HULC are important for maintaining the mesenchymal phenotype in this cell line. Genomic analysis showed an inverse correlation between expression of genes after knockdown of HULC and expression of those genes in liver tumors from patients. The antidiabetic drug metformin down-regulates Sp proteins in pancreatic cancer, and similar results including decreased HULC expression were observed in HepG2, SNU-449 and SK-Hep-1 cells treated with metformin, indicating that metformin and other antineoplastic agents that target Sp proteins may have clinical applications for HCC chemotherapy.