Knockdown Of Sp1 Research Articles

Sp1 trans-activates and is required for maximal aldosterone induction of the αENaC gene in collecting duct cells

The epithelial Na+ channel (ENaC) in the distal nephron constitutes the rate-limiting step for renal sodium reabsorption. Aldosterone increases tubular sodium absorption in large part by increasing αENaC transcription in collecting duct principal cells. We previously reported that Af9 binds to +78/+92 of αENaC and recruits Dot1a to repress basal and aldosterone-sensitive αENaC transcription in mouse inner medullary collecting duct (mIMCD)3 cells. Despite this epigenetic repression, basal αENaC transcription is still evident and physiologically necessary, indicating basal operation of positive regulators. In the present study, we identified Sp1 as one such regulator. Gel shift and antibody competition assays using a +208/+240 probe revealed DNA-Sp1-containing complexes in mIMCD3 cells. Mutation of the +222/+229 element abrogated Sp1 binding in vitro and in promoter-reporter constructs stably expressed in mIMCD3 cells. Compared with the wild-type promoter, an αENaC promoter-luciferase construct with +222/+229 mutations exhibited much lower activity and impaired trans-activation in Sp1 overexpression experiments. Conversely, Sp1 knockdown inhibited endogenous αENaC mRNA and the activity of the wild-type αENaC promoter but not the mutated construct. Aldosterone triggered Sp1 recruitment to the αENaC promoter, which was required for maximal induction of αENaC promoter activity and was blocked by spironolactone. Sequential chromatin immunoprecipitation assays and functional tests of +78/+92 and +222/+229 αENaC promoter mutants indicated that while Sp1, Dot1a, and Af9 co-occupy the αENaC promoter, the Sp1 effects are functionally independent from Dot1a and Af9. In summary, Sp1 binding to a cis-element at +222/+229 represents the first identified constitutive driver of αENaC transcription, and it contributes to maximal aldosterone trans-activation of αENaC.

Sp1 mediates microRNA-29c-regulated type I collagen production in renal tubular epithelial cells

Specificity protein 1 (Sp1), a ubiquitously expressed transcription factor, plays a potential pathogenic role for fibrotic disease in many organs by regulating the expression of several fibrosis-related genes, however, its role in kidney fibrosis and the mechanisms regulating its expression remain incompletely clarified. Here, we found that Sp1 was markedly induced and closely correlated with interstitial type I collagen accumulation in kidney tubular epithelia from obstructive nephropathy. In vitro, both Sp1 and type I collagen expression were up-regulated in TGF-β1-treated kidney tubular epithelial cells (NRK-52E), whereas knockdown of Sp1 largely abolished TGF-β1-induced type I collagen production, suggesting that Sp1 induction is partially responsible for type I collagen expression. In addition, we found that miR-29c expression was remarkably reduced in either the tubular epithelial cells from kidney with UUO nephropathy or TGF-β1-treated NRK-52E cells. Knockdown of miR-29c could sufficiently induce Sp1 and type I collagen expression, whereas ectopic expression of miR-29c largely abolished their expression stimulated by TGF-β1 in NRK-52E cells. Furthermore, knockdown of Sp1 effectively hindered type I collagen induction stimulated by miR-29c down-regulation. Collectively, this study demonstrates that Sp1 acts as an essential mediator for miR-29c in regulating type I collagen production in tubular epithelial cells, which may provide a novel mechanistic insight about miR-29c in renal fibrosis.

Critical Role of Histone Acetylation by p300 in Human Placental 11β-HSD2 Expression

Fetal overexposure to glucocorticoids leads to growth restriction. Optimal fetal glucocorticoid level is ensured by the expression of cortisol-inactivating enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) in placental syncytiotrophoblasts. The transcription factor selective promoter factor 1 (Sp1) is known to up-regulate 11β-HSD2 expression in the presence of enhanced histone acetylation in syncytiotrophoblasts, but the mechanisms underlying histone acetylation remain unknown. The role of p300 in histone acetylation associated with 11β-HSD2 expression in syncytiotrophoblasts was investigated. Distribution of p300 in human placenta was studied with immunohistochemistry. The role of p300 in histone-3 (H3) acetylation in association with 11β-HSD2 expression was investigated in cultured primary human placental trophoblasts in the presence of small interfering RNA (siRNA)-mediated knockdown of p300, p300 inhibitor C646, or p300 overexpression. The interaction of Sp1 and p300 was studied with chromatin immunoprecipitation and coimmunoprecipitation. Intense staining of p300 was found in the nuclei of trophoblasts. Levels of p300 and acetyl H3K9 and H3K27 associated with 11β-HSD2 promoter were increased in the course of syncytialization and by cAMP pathway activation. Chromatin immunoprecipitation and coimmunoprecipitation revealed p300 and Sp1 on 11β-HSD2 promoter and in the same protein complex in the syncytiotrophoblasts. Overexpression of p300 enhanced 11β-HSD2 expression, which was attenuated by Sp1 knockdown, whereas p300 knockdown and C646 reduced both basal and cAMP-stimulated acetylation of H3K9 and H3K27 associated with 11β-HSD2 expression. Interaction of p300 with Sp1 plays a crucial role in histone acetylation associated with 11β-HSD2 expression in syncytiotrophoblasts, which may have important implications in the establishment of the placental glucocorticoid barrier in gestation.

S100P/RAGE signaling regulates microRNA-155 expression via AP-1 activation in colon cancer

Accumulating evidence indicates that elevated S100P promotes the pathogenesis of cancers, including colon cancer. S100P exerts its effects by binding to and activating the Receptor for Advance Glycation End-products (RAGE). The effects of up-regulated S100P/RAGE signaling on cell functions are well documented. Despite these observations, little is known about the downstream targets of S100P/RAGE signaling. In the present study, we demonstrated for the first time that activation of RAGE by S100P regulates oncogenic microRNA-155 (miR-155) expression through Activator Protein-1 (AP-1) stimulation in colon cancer cells. Ectopic S100P up-regulated miR-155 levels in human colon cancer cells. Conversely, knockdown of S100P resulted in a decrease in miR-155 levels. Exogenous S100P induced miR-155 expression, but blockage of the RAGE with anti-RAGE antibody suppressed the induction of miR-155 by exogenous S100P. Attenuation of AP-1 activation through pharmacological inhibition of MEK activation or genetic inhibition of c-Jun activation using dominant negative c-Jun (TAM67) suppressed miR-155 induction by exogenous S100P. Also, S100P treatment stimulated the enrichment of c-Fos, an AP-1 family member, at the miR-155 host gene promoter site. Finally, a functional study demonstrated that miR-155 knockdown decreases colon cancer cell growth, motility, and invasion. Altogether, these data demonstrate that the expression of miR-155 is regulated by S100P and is dependent on RAGE activation and stimulation of AP-1.

Abstract 1806: AP-1 transcriptionally regulates expression of miR-155 in colon cancer cells.

Abstract Accumulating evidence indicates elevated S100P promotes the pathogenesis of cancers, including colon cancer. S100P exerts its effects by binding to and activating the Receptor for Advance Glycation End-products (RAGE). The effects of up-regulated S100P/RAGE signaling on cell functions are well documented. Despite these overwhelming evidences, little is known about the downstream targets of S100P/RAGE signaling. In the present study, we demonstrated for the first time that activation of RAGE by S100P regulates oncogenic microRNA-155 (miR-155) expression through Activator Protein-1 (AP-1) stimulation in colon cancer cells. Both S100P and miR-155 expressions are up-regulated in colon tumor specimens. Ectopic S100P expression leads to elevation of miR-155 level. Conversely, knockdown of S100P results in a decrease in miR-155 levels. Exogenous S100P induces miR-155 expression, but blockage of the RAGE receptor with anti-RAGE antibody suppresses the induction of miR-155 by exogenous S100P. Attenuation Blockage of AP-1 activation by S100P, through pharmacological inhibition of MEK activation or genetic inhibition of c-Jun activation using dominant negative c-Jun (TAM67) suppresses miR-155 induction by exogenous S100P. Exogenous S100P treatment stimulates the enrichment of c-Fos, an AP-1 family member at the miR-155 promoter site. Finally, functional study shows that miR-155 knockdown decreases colon cancer cell cell cell growtholon formation and motilityotility in S100P stably transfected cells and parental cells. Taken together, these data demonstrate that the expression of miR-155 is regulated by S100P is dependent on RAGE activation and stimulation of AP-1. Furthermore, the results show that miR-155 is a downstream target of S100P/RAGE signaling and a critical player in S100P functions in colon cancer cells. Citation Format: Benjamin C. Onyeagucha, Melania Mercado-Pimentel, Erik Flemington, Mark A. Nelson. AP-1 transcriptionally regulates expression of miR-155 in colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1806. doi:10.1158/1538-7445.AM2013-1806

Regulation of the Na+,HCO3‐ cotransporter NBCn1 (SLC4A7) by a constitutively active ErbB2 receptor in MCF‐7 breast cancer cells

Net acid production is greatly increased in tumors due to a shift towards glycolytic metabolism. We previously showed that the Na+,HCO3− cotransporter NBCn1 (SLC4A7) is strongly upregulated in MCF‐7 breast cancer cells upon expression of a truncated, constitutively active ErbB2 receptor, ΔNErbB2. Here, we explore the mechanisms involved in this upregulation. NBCn1 mRNA and protein levels were reduced by inhibition of ERK or Src in ΔNErbB2‐, but not vector‐expressing MCF‐7 cells. PI3K‐Akt inhibition reduced NBCn1 expression in both cell types. NBCn1 expression was increased by stimulation of full‐length ErbB receptors. In luciferase promoter assays, the 1.2 kb genomic region upstream of SLC4A7 increased luciferase activity after ΔNErbB2 expression ~2.5 fold. Promoter deletions identified the ΔNErbB2‐ responsive region and putative transcription factor binding sites. NBCn1 expression was increased by expression of dominant‐negative (DN)‐Sp1 or by Sp1 knockdown, and decreased by knockdown of Krüppel‐like factor 4 (KLF4), while DN‐Sp3 or Sp3 knockdown had no effect. Chromatin immunoprecipitation assay (ChIP) demonstrated Sp1, Sp3 and KLF4 binding to the NBCn1 promoter. In conclusion, we identified an NBCn1 promoter and demonstrated that NBCn1 transcriptional regulation involves Sp1 and KLF4, and that ErbB2‐dependent upregulation of NBCn1 is controlled at least in part by ERK‐ and Src signaling.Funding: Danish Cancer Society, Danish Research Council

MiR-22 is down-regulated in gastric cancer, and its overexpression inhibits cell migration and invasion via targeting transcription factor Sp1

Accumulating evidence has shown that microRNAs are involved in multiple processes in cancer development and progression. Recently, miR-22 has been identified as a tumor-suppressing microRNA in many human cancers. However, the specific function of miR-22 in gastric cancer is unclear at this point. In this study, we first measured miR-22 expression level in 30 pairs of gastric cancer and matched normal tissues, two normal and six gastric cancer cell lines by real-time quantitative RT-PCR. We found that the expression of miR-22 in gastric cancer tissues and cell lines was much lower than that in normal control, respectively. Transfection of miR-22 expression plasmid could significantly inhibit the cell migration and invasion in SGC-7901 and NCL-N87 gastric cancer cell lines. Moreover, we also showed that Sp1 was negatively regulated by miR-22 at the posttranscriptional level, via a specific target site within the 3'UTR by luciferase reporter assay. The expression of Sp1 was inversely correlated with miR-22 expression in gastric cancer tissues, and knockdown of Sp1 by siRNA inhibited cell malignant behaviors. Thus, our findings suggest that miR-22 acts as tumor suppressor by targeting the Sp1 gene and inhibiting gastric cancer cell migration and invasion. The findings of this study contribute to current understanding of the functions of miR-22 in gastric cancer.

Cloning and characterization of the human USP22 gene promoter.

Ubiquitin-specific processing enzyme 22 (USP22) plays a direct role in regulating cell cycle, and its overexpression has been reported to be involved in tumor progression. However, little is known about the regulation of USP22 transcription. In this study, we cloned and characterized the human USP22 promoter. Using 5′ RACE (rapid amplification of cDNA ends) analysis, the transcriptional initiation site was identified. Promoter deletion analysis showed that the sequence between −210 and −7 contains the basal promoter for USP22 in human fibroblast and tumor cells. Surprisingly, mutations in a putative Sp1 binding site immediately upstream of the USP22 transcriptional start site (−13 to −7) resulted in a significant induction of promoter activity. Further study revealed that Sp1 binds to this site in human normal fibroblast cells, and treatment with the Sp1 inhibitor mithramycin A led to a marked increase in USP22 transcript levels. Forced expression of exogenous Sp1 repressed the USP22 promoter activity in HeLa cells. In contrast, knockdown of Sp1 enhanced USP22 promoter activity and mRNA levels. These data suggest that Sp1 is a crucial regulator of USP22 transcription.

Prostaglandin E2 induces stromal cell-derived factor-1 expression in prostate stromal cells by activating protein kinase A and transcription factor Sp1

Recent reports indicate prostaglandin E2 (PGE2) can modulate tumor environment and promote angiogenesis through induction of stromal cell-derived factor 1 (SDF-1) production. We investigated the mechanism of PGE2-induced SDF-1 regulation in human prostate stromal cell and analyzed the effects in a stromal–epithelial interaction model. PGE2 stimulation increased SDF-1 expression in the prostate stromal cell lines WPMY-1 and NAF. We revealed signaling through the PGE2 receptor EP3 and activation of protein kinase A (PKA) are required. The EP3 agonist sulprostone and the cAMP analog forskolin mimicked and the EP3 siRNA, antagonist L798106 and the PKA inhibitor H89 abrogated the effect of PGE2 on SDF-1 expression. SDF-1 promoter truncation experiments demonstrated a 254bp (from nt −219 to nt +34) SDF-1 proximal promoter fragment containing 5 putative transcription factor Sp1 binding motifs is sufficient for PGE2 induction. CHIP assays confirmed binding and PGE2 induced recruitment of Sp1 to the SDF-1 promoter. Sp1 motif mutation identified Sp1 motifs −140/−133 and −9/+1 as the crucial elements responsible for PGE2 induction. Moreover, SDF-1 was up- or down-regulated by Sp1 over-expression or knock-down. We also demonstrate stimulation of migration of prostate cancer cell lines PC3 and DU145 with conditioned media collected from WPMY-1 or NAF cells stimulated with PGE2 and blockade of enhanced migration by a SDF-1 neutralizing antibody. In conclusion, we provide evidence for a paracrine prostate stromal–epithelial interaction induced by upregulation of expression of SDF-1 by PGE2. Our research provides new insights into the mechanism promoting metastasis of prostate carcinoma via stromal–epithelial interaction.

Mitochondria-Localized Glutamic Acid-Rich Protein (MGARP) Gene Transcription Is Regulated by Sp1

BackgroundMitochondria-localized glutamic acid-rich protein (MGARP) is a novel mitochondrial transmembrane protein expressed mainly in steroidogenic tissues and in the visual system. Previous studies showed that MGARP functions in hormone biosynthesis and its expression is modulated by the HPG axis.Methodology/Principal FindingsBy bioinformatics, we identified two characteristic GC-rich motifs that are located proximal to the transcription start site (TSS) of MGARP, and each contains two Specificity protein 1 (Sp1) binding elements. We then determined that the −3 kb proximal MGARP promoter is activated in a Sp1-dependent manner using reporter assays and knockdown of Sp1 led to decreased expression of endogenous MGARP messages. We also demonstrated that one of the two GC-rich motifs, GC-Box1, harbors prominent promoter activity mediated by Sp1, and that it requires both GC boxes for full transcriptional activation. These findings suggest a dominant role for these GC boxes and Sp1 in activating the MGARP promoter through a synergistic mechanism. Consistently, the results of an Electrophoretic Mobility Gel Shift Assay (EMSA) and Chromatin Immunoprecipitation (ChIP) confirmed that Sp1 specifically interacts with the GC-rich region. We further found that estrogen receptor α (ERα), a known Sp1 co-activator, could potentiate GC-boxes containing MGARP promoter activity and this effect is mediated by Sp1. Knockdown of Sp1 significantly diminished the MGARP promoter transactivation and the expression of endogenous MGARP mediated by both Sp1 and ERα.Conclusions/SignificanceThe present study identified a proximal core sequence in the MGARP promoter that is composed of two enriched Sp1 binding motifs and established Sp1 as one major MGARP transactivator whose functions are synergistic with ERα, providing a novel understanding of the mechanisms of MGARP gene transcriptional regulation.

Acetylated Sp1 inhibits PTEN expression through binding to PTEN core promoter and recruitment of HDAC1 and promotes cancer cell migration and invasion

Specificity protein 1 (Sp1) is often overexpressed in cancer cells. Its binding sites are known to exist in the phosphatase and tension homolog deleted on chromosome 10 (PTEN) promoter. In this study, we hypothesized that Sp1 negatively regulates PTEN expression. We used several cell lines to determine the effects of Sp1. The results showed that Sp1 overexpression inhibited the expression and promoter activity of PTEN and correspondingly upregulated AKT phosphorylation, whereas Sp1 knockdown upregulated the expression and promoter ability of PTEN and downregulated AKT phosphorylation. Moreover, a series of deletion and site-directed mutations of the PTEN promoter indicated that Sp1 can inhibit PTEN promoter activity through a specific Sp1-binding site at the PTEN core promoter in vivo. Meanwhile, non-acetylated Sp1, with its loss of DNA binding activity, failed to inhibit the expression and promoter activity of PTEN. Histone deacetylase 1 was necessary for Sp1 to inhibit PTEN expression. The inverse expression of Sp1 and PTEN was found in tongue cancer cells and salivary adenoid cystic cancer (SACC)-LM cells (possessing higher potential for lung metastasis than SACC-83) as compared with that in adjacent normal tissue and SACC-83 cells, respectively. Sp1 knockdown decreased the migration and invasion of SACC-LM cells, whereas Sp1 overexpression increased the migration and invasion of SACC-83 cells. Overall, these results suggest that Sp1 is involved in the development and invasiveness of cancer through inhibition of PTEN.

Cyclooxygenase‐2 up‐regulates CCR7 expression via AKT‐mediated phosphorylation and activation of Sp1 in breast cancer cells

Up-regulation of cyclooxygenase-2 (COX-2) is frequently found in human cancers and is significantly associated with tumor metastasis. Our previous results demonstrate that COX-2 and its metabolite prostaglandin E2 (PGE2) stimulate the expression of CCR7 chemokine receptor via EP2/EP4 receptors to promote lymphatic invasion in breast cancer cells. In this study, we address the underlying mechanism of COX-2/PGE2-induced CCR7 expression. We find that COX-2/PGE2 increase CCR7 expression via the AKT signaling pathway in breast cancer cells. Promoter deletion and mutation assays identify the Sp1 site located at the -60/-57 region of CCR7 gene promoter is critical for stimulation. Chromatin immunoprecipitation (ChIP) assay confirms that in vivo binding of Sp1 to human CCR7 promoter is increased by COX-2 and PGE2. Knockdown of Sp1 by shRNA reduces the induction of CCR7 by PGE2. We demonstrate for the first time that AKT may directly phosphorylate Sp1 at S42, T679, and S698. Phosphorylation-mimic Sp1 protein harboring S42D, T679D, and S698D mutation strongly activates CCR7 expression. In contrast, change of these three residues to alanine completely blocks the induction of CCR7 by PGE2. Pathological investigation demonstrates that CCR7 expression is strongly associated with phospho-AKT and Sp1 in 120 breast cancer tissues. Collectively, our results demonstrate that COX-2 up-regulates CCR7 expression via AKT-mediated phosphorylation and activation of Sp1 and this pathway is highly activated in metastatic breast cancer.

The transcription factor Sp1 is responsible for aging‐dependent altered nucleocytoplasmic trafficking

Hyporesponsiveness to external signals, such as growth factors and apoptotic stimuli, is a cardinal feature of cellular senescence. We previously reported that an aging-dependent marked reduction in nucleocytoplasmic trafficking (NCT)-related genes could be responsible for this phenomenon. In searching for the mechanism, we identified the transcription factor, Sp1, as a common regulator of NCT genes, including various nucleoporins, importins, exportins, and Ran GTPase cycle-related genes. Sp1 knockdown led to a reduction of those genes in young human diploid fibroblast cells (HDF); Sp1 overexpression induced those genes in senescent cells. In addition, epidermal growth factor stimulation-induced p-ERK1/2 nuclear translocation and Elk-1 phosphorylation were severely impaired by Sp1 depletion in young HDFs; Sp1 overexpression restored the nuclear translocation of p-ERK1/2 in senescent HDFs. Furthermore, we observed that Sp1 protein levels were decreased in senescent cells, and H(2) O(2) treatment decreased Sp1 levels in a proteasome-dependent manner. In addition, O-GlcNAcylation of Sp1 was decreased in senescent cells as well as in H(2) O(2) -treated cells. Taken together, these results suggest that Sp1 could be a key regulator in the control of NCT genes and that reactive oxygen species-mediated alteration in Sp1 stability may be responsible for the generalized repression of those genes, leading to formation of the senescence-dependent functional nuclear barrier, resulting in subsequent hyporesponsiveness to external signals.

A possible gene silencing mechanism: Hypermethylation of the Keap1 promoter abrogates binding of the transcription factor Sp1 in lung cancer cells

Hypermethylation often leads to gene silencing; however, the mechanism responsible for the low expression resulting from hypermethylation of the tumor suppressor gene Kelch-like ECH-associating protein 1 (Keap1) in human lung cancer cell lines remains unclear. In this study, using promoter deletion and site mutagenesis assays, we determined that one transcription factor stimulating protein-1 (Sp1) regulatory element in the Keap1 promoter region was important for the transcription of Keap1 in A549 cells. We demonstrated that the transcription factor Sp1 can directly bind to this element in the normal bronchial epithelial BEAS-2B cell line but not in A549 cells, as assessed with chromatin immunoprecipitation (ChIP). EMSAs and supershift assays also showed that CpG island methylation could abrogate Sp1 binding to the Keap1 promoter. Moreover, Keap1 mRNA decreased by 50% after the knock-down of Sp1 with siRNA in BEAS-2B cells, whereas the over-expression of Sp1 led to a dramatic increase in Keap1 promoter activity. The treatment of A549 cells with 5-aza-2′-deoxycytidine restored the binding of Sp1 to the promoter and Keap1 expression. Our results indicate that Sp1 is essential for Keap1 expression and that promoter methylation blocks Sp1 binding in A549 cells. These results demonstrate that hypermethylation may act as an epigenetic gene silencing mechanism, i.e., the inhibition of Sp1 binding to the hypermethylated Keap1 promoter in lung cancer cells, which suggests new approaches to lung cancer treatment.

Betulinic Acid Decreases Specificity Protein 1 (Sp1) Level via Increasing the Sumoylation of Sp1 to Inhibit Lung Cancer Growth

Previous studies have shown that the inhibitory effect of betulinic acid (BA) on specificity protein 1 (Sp1) expression is involved in the prevention of cancer progression, but the mechanism of this effect remains to be delineated. In this study, we determined that BA treatment in HeLa cells increased the sumoylation of Sp1 by inhibiting sentrin-specific protease 1 expression. The subsequent recruitment of E3 ubiquitin-protein ligase RING finger protein 4 resulted in ubiquitin-mediated degradation in a 26S-proteosome-dependent pathway. In addition, both BA treatment and mithramycin A (MMA) treatment inhibited lung tumor growth and down-regulated Sp1 protein expression in Kras(G12D)-induced lung cancers of bitransgenic mice. In gene expression profiles of Kras(G12D)-induced lung cancers in bitransgenic mice with and without Sp1 inhibition, 542 genes were affected by MMA treatment. One of the gene products, cyclin A2, which was involved in the S and G(2)/M phase transition during cell cycle progression, was investigated in detail because its expression was regulated by Sp1. The down-regulation of cyclin A2 by BA treatment resulted in decreased retinoblastoma protein phosphorylation and cell cycle G(2)/M arrest. The BA-mediated cellular Sp1 degradation and antitumor effect were also confirmed in a xenograft mouse model by using H1299 cells. The knockdown of Sp1 in lung cancer cells attenuated the tumor-suppressive effect of BA. Taken together, the results of this study clarify the mechanism of BA-mediated Sp1 degradation and identify a pivotal role for Sp1 in the BA-induced repression of lung cancer growth.

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.

Betulinic Acid Targets YY1 and ErbB2 through Cannabinoid Receptor-Dependent Disruption of MicroRNA-27a:ZBTB10 in Breast Cancer

Treatment of ErbB2-overexpressing BT474 and MDA-MB-453 breast cancer cells with 1 to 10 μmol/L betulinic acid inhibited cell growth, induced apoptosis, downregulated specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and decreased expression of ErbB2. Individual or combined knockdown of Sp1, Sp3, Sp4 by RNA interference also decreased expression of ErbB2 and this response was because of repression of YY1, an Sp-regulated gene. Betulinic acid-dependent repression of Sp1, Sp3, Sp4, and Sp-regulated genes was due, in part, to induction of the Sp repressor ZBTB10 and downregulation of microRNA-27a (miR-27a), which constitutively inhibits ZBTB10 expression, and we show for the first time that the effects of betulinic acid on the miR-27a:ZBTB10-Sp transcription factor axis were cannabinoid 1 (CB1) and CB2 receptor-dependent, thus identifying a new cellular target for this anticancer agent.

Sp1 and Sp3 are involved in the full transcriptional activity of centromere protein H in human nasopharyngeal carcinoma cells

The overexpression of centromere protein H (CENPH), one of the fundamental components of the human active kinetochore, has been shown to be closely associated with human cancers. However, the mechanism of its transcriptional regulation has not been reported. The aim of the present study was to investigate the regulatory elements for the transcriptional regulation of CENPH in nasopharyngeal carcinoma cells. To characterize the CENPH promoter and identify regulatory elements, we cloned 1015 bp (-975/+40 bp) of the 5'-flanking region of the CENPH gene from immortalized normal nasopharyngeal epithelial cells (Bmi-1/NPEC). Functional analysis established a minimal region (-140/-87 bp) involved in the regulation of human CENPH promoter activity. Through site-directed mutagenesis, a transactivation assay, chromatin immunoprecipitation, and electrophoretic mobility shift assay, we found that the Sp1/Sp3 transcription factors could bind to the CENPH promoter in vitro and in vivo, and that they regulated CENPH promoter activation in human nasopharyngeal carcinoma cells. Furthermore, Sp1 and Sp3 were highly expressed in nasopharyngeal carcinoma cells. Knockdown of Sp1 and Sp3 by small interfering RNA or inhibition of Sp1 and Sp3 activity by mithramycin A decreased CENPH mRNA expression, whereas the exogenous expression of Sp1 and Sp3 upregulated CENPH mRNA expression. Taken together, our results indicate that Sp1 and Sp3 bind to the CENPH minimal promoter and function as a regulator of the transcription of CENPH in human nasopharyngeal carcinomas.

Abstract 2211: Accessible chromatin structure permits factors Sp1 and Sp3 to regulate human TGFBI gene expression

Abstract Transforming growth factor beta 1-induced (TGFBI) protein is an extracellular matrix (ECM) protein expressed widely in human tissues. The gene encoding this protein was first described as a major TGF-b-responsive gene in the lung adenocarcinoma cell line A549. TGFBI appears to mediate cell adhesion and migration through interaction with integrin, and increased TGFBI expression has been associated with inflammatory diseases including rheumatoid arthritis, atherosclerosis, cyclosporine nephropathy, and stab wound healing. TGFBI is ubiquitously expressed in healthy tissues, while disrupted TGFBI expression has been described in human tumor cell lines and in human primary tumor samples. A general function of this protein appears to be as a promoter or suppressor of cancer progression, depending on the tissue. Regulation of TGFBI expression by epigenetic mechanism such as DNA methylation and microRNA has been reported, but few studies have focused on how chromatin structure affects transcriptional activity relating to TGFBI expression. We show that both chromatin structure and regulatory elements in the TGFBI promoter region that bind factors Sp1 and Sp3 regulate constitutive expression of TGFBI. In this study, we investigated how human TGFBI expression is regulated in lung and breast cancer cells. We observed that the TGFBI promoter in A549 and MBA-MD-231 cells, which constitutively express TGFBI, existed in an open chromatin conformation associated with transcriptionally permissive histone modifications. Moreover, we found that TGFBI expression required Sp1 transcription elements that can bind transcription factors Sp1 and Sp3 in vitro. Occupancy of the TGFBI promoter by Sp1 and Sp3 in vivo was only observed in TGFBI-expressing cells, indicating that open chromatin conformation might facilitate the binding of Sp1 and Sp3 to the TGFBI promoter region. TGFBI promoter activity was impaired when Sp1 elements were mutated, but was increased when Sp1 or Sp3 factors was overexpressed. Furthermore, Sp1 inhibition in vivo by mithramycin A, as well as knockdown of Sp1 and/or Sp3 expression by short interfering RNA, significantly reduced TGFBI mRNA and protein levels. Thus, our data demonstrated that the expression of TGFBI is well correlated with chromatin conformation at the TGFBI promoter, and that factors Sp1 and Sp3 are the primary determinants for the control of constitutive expression of TGFBI gene. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2211. doi:1538-7445.AM2012-2211

Abstract CT-06: The combination of bortezomib (BOR) and decitabine (DEC): A phase I trial in patients (pts) with acute myeloid leukemia (AML) targeting FLT3 expression

Abstract Most pts with AML are not cured with conventional chemotherapy regimens. Gain of function mutations & overexpression of tyrosine kinase (TK) receptors (ie KIT & FLT3) are associated with leukemogenesis and poor outcome & represent therapeutic targets. The use of TK inhibitors in AML has been disappointing. Recently we showed that BOR downregulates KIT in AML cells by upregulating miR-29b that disrupts a KIT transactivating complex comprising SP1 and NFκB (p65). In silico we found SP1 & p65 binding sites also in the FLT3 promoter region & speculated that FLT3 may be regulated similarly. Utilizing chromatin immunoprecipitation (CHIP) in MV4-11 cells expressing high FLT3 levels we showed enrichment of SP1 (1.6 fold) & p65 (3.1 fold) vs IgG control on the FLT3 promoter. Cotransfection of a luciferase reporter harboring the FLT3 promoter with SP1 or p65 vectors in 293T cells increased luciferase activity (3.1 & 3.0. fold); siRNA mediated knock-down of SP1 or p65 decreased activity (0.5 & 0.7 fold). In KG1 cells with low FLT3 levels, overexpression of SP1 or p65 increased FLT3 (1.7 & 1.6 fold); in MV4-11 cells siRNA-mediated knockdown of SP1 or p65 decreased FLT3 (0.05 & 0.3 fold) mRNA & protein. Since miR-29b can disrupt the SP1/p65 complex we overexpressed miR-29b in MV4-11 cells, which consequently downregulated FLT3 mRNA & protein level in MV4-11 cells to barely detectable levels. Both BOR & DEC can upregulate miR-29b; thus these drugs potentially decrease FLT3. Indeed BOR (60nM at 24h) or DEC (2.5µM at 48h) led to decreased FLT3 in MV4-11 cells (<.001 & 0.5 fold) & in primary AML blasts (0.28 & 0.42 fold). We concluded that BOR & DEC target the expression of FLT3 via upregulation of miR-29b & disruption of the SP1/p65 complex. To validate these results, we assessed the expression of FLT3 in AML pts enrolled on a Phase I clinical trial (NCT00703300) that tested the BOR & DEC combination. Pts with relapsed/refractory AML or age >60 years with previously untreated disease received DEC at 20mg/m2 IV daily on days 1-10. BOR was gradually dose escalated to 1.3 mg/m2 given on days 5, 8, 12 & 15. Cycles were repeated every 28 days. Pretreatment and day 26 expression levels of FLT3 and miR-29b were assessed. 19 patients were enrolled with a median age of 69 years (range: 32-83). Pts received a median of 2 cycles of therapy (range, 1-14 cycles). Febrile neutropenia & infectious complications were frequent. BOR & DEC at target doses were tolerable; neuropathy following repetitive cycles of BOR limited its administration. 7 pts achieved disease remission. Consistent with our preclinical results, miR-29b expression increased (median fold change: 3.9) and FLT3 expression levels decreased (median fold change: 0.4). As the FLT3 targeting results are encouraging a phase II study of BOR & DEC combination is being planned with modifications of BOR dosing to avoid neuropathy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr CT-06. doi:1538-7445.AM2012-CT-06