Factor Sp1 Research Articles

Changes in the Expression of Transcription Factors Involved in Modulating the Expression of EPO-R in Activated Human CD4-Positive Lymphocytes

We have recently described the presence of the erythropoietin receptor (EPO-R) on CD4+ lymphocytes and demonstrated that its expression increases during their activation, reaching a level reported to be typical for erythroid progenitors. This observation suggests that EPO-R expression is modulated during lymphocyte activation, which may be important for the cells’ function. Here we investigated whether the expression of GATA1, GATA3 and Sp1 transcription factors is correlated with the expression of EPO-R in human CD4+ lymphocytes stimulated with monoclonal anti-CD3 antibody. The expression of GATA1, GATA3 and Sp1 transcription factors in CD4+ cells was estimated before and after stimulation with anti-CD3 antibody by Western Blot and flow cytometry. The expression of EPO-R was measured using real-time PCR and flow cytometry. There was no change in the expression of GATA1 and GATA3 in CD4+ lymphocytes after stimulation with anti-CD3 antibody. However, stimulation resulted in the significantly increased expression of the Sp1 factor. CD4+ lymphocytes stimulated with anti-CD3 antibody exhibited an increase in both the expression level of EPOR gene and the number of EPO-R molecules on the cells’ surface, the latter being significantly correlated with the increased expression of Sp1. Sp1 is noted to be the single transcription factor among the ones studied whose level changes as a result of CD4+ lymphocyte stimulation. It seems that Sp1 may significantly affect the number of EPO-R molecules present on the surface of activated CD4+ lymphocytes.

AOSOP14 MATRIX-PRODUCING CARCINOMA OF THE BREAST: IMMUNOHISTOCHEMICAL EXPRESSION PROFILES OF A RARE SUBTYPE OF BREAST CARCINOMA

Introduction. Matrix-producing carcinoma (MPC) of the breast is a rare subtype of breast carcinoma. As a rare entity, clinicopathological features and its response to therapy are still unclear. To better understand the biological features of MPC, we investigated its immunohistochemical expression profiles. Methods. We undertook immunohistochemical study for oestrogen receptor (ER),progesterone receptor (PR),HER2,Ki67, cytokeratin (CK) 5, CK14, epidermal growth factor receptor (EGFR), androgen receptor (AR), p53, c-MET, and laminin on 11 cases of MPC. Results. All cases were triple negative breast cancer. Six cases showed a high Ki67 score (>50%). Most cases were immunoreactive for CK5 (10/11), CK14 (9/11), and EGFR (10/11). Three cases were positive for AR. Nine cases were positive for p53. Positive immunoexpression of c-MET was observed in one case, and of laminin in ten cases. The one metastatic lymph node showed the same matrix-producing pattern but expressed CK5, EGFR, and Ki67 differently from the primary site. Discussion. These data suggest that MPC has the immunohistochemical features of basal-like breast carcinoma, and these expression profiles might be helpful in biomarker selection for appropriate targeted therapies. Funding. None declared. The authors declared no conflicts of interest.

AOSOP15 PHASE 2 STUDY COMPARING CISPLATIN/ETOPOSIDE AND WEEKLY PACLITAXEL/CARBOPLATIN REGIMENS WITH CONCURRENT THORACIC RADIOTHERAPY IN PATIENTS WITH LOCALLY ADVANCED NON-SMALL-CELL LUNG CANCER

Introduction. Matrix-producing carcinoma (MPC) of the breast is a rare subtype of breast carcinoma. As a rare entity, clinicopathological features and its response to therapy are still unclear. To better understand the biological features of MPC, we investigated its immunohistochemical expression profiles. Methods. We undertook immunohistochemical study for oestrogen receptor (ER),progesterone receptor (PR),HER2,Ki67, cytokeratin (CK) 5, CK14, epidermal growth factor receptor (EGFR), androgen receptor (AR), p53, c-MET, and laminin on 11 cases of MPC. Results. All cases were triple negative breast cancer. Six cases showed a high Ki67 score (>50%). Most cases were immunoreactive for CK5 (10/11), CK14 (9/11), and EGFR (10/11). Three cases were positive for AR. Nine cases were positive for p53. Positive immunoexpression of c-MET was observed in one case, and of laminin in ten cases. The one metastatic lymph node showed the same matrix-producing pattern but expressed CK5, EGFR, and Ki67 differently from the primary site. Discussion. These data suggest that MPC has the immunohistochemical features of basal-like breast carcinoma, and these expression profiles might be helpful in biomarker selection for appropriate targeted therapies. Funding. None declared. The authors declared no conflicts of interest.

Circulation Research “In This Issue” Anthology

<i>Circulation Research</i> “In This Issue” Anthology

Effects of dihydromyricetin on tumor necrosis factor and NF-κB p65 of RAU rats

Effects of dihydromyricetin on tumor necrosis factor and NF-κB p65 of RAU rats

The role of NF-κB activation during protection against Leishmania infection

Members of the nuclear factor-κB (NF-κB) family of transcription factors regulate a variety of molecules involved in host defense against pathogens. A prominent role of NF-κB in innate and adoptive immunity is based on the regulation of inducible transcription of various genes whose products are essential components of the immune response such as cytokines, chemokines, and adhesion molecules. Since the discovery of the five members of the NF-κB transcription factor family, RelA, c-Rel, RelB, p50 and p52, considerable progress has been made toward better understanding how the different NF-κB homo- and heterodimers regulate such distinct subsets of target genes. All of the NF-κB molecules are activated by various infectious stimuli; however, there are still open questions related to the selective functions of individual NF-κB family members during a coordinated immune response to infection. Diverse parasites such as Toxoplasma gondii, Leishmania donovani, Leishmania major, and Trichuris muris have been reported to activate NF-κB signaling cascades, and a number of distinct parasite-derived molecules may actively interfere with the pathways that lead to NF-κB activation. In this review, we provide an overview on the role of NF-κB activation in leishmaniasis and discuss how individual NF-κB family members might perform their distinct and non-overlapping functions in the regulation of protective immunity to Leishmania infection.

P68 RNA helicase promotes glioma cell proliferation in vitro and in vivo via direct regulation of NF- B transcription factor p50

The DEAD-box RNA helicase p68 plays a very important role in early organ development and maturation. However, the role of p68 in glioma is unclear. In this study, we showed that p68 protein levels were significantly elevated in high-grade gliomas compared to low-grade gliomas and normal adjacent brain tissues. Importantly, the expression of p68 was significantly associated with poorer overall survival and enhanced resistance to treatment with radiotherapy plus temozolomide for glioma patients. Ectopic expression of p68 enhanced glioma cell proliferation both in vitro and in vivo. In contrast, knockdown of endogenous p68 prevented glioma cell proliferation. Using a tandem affinity purification assay, we found a new p68-binding protein, nuclear factor (NF)-κB p50. We found that p68 bound with the N-terminal of NF-κB p50, and the mutant of p68 lacking the p50-interaction domain failed to stimulate glioma cell proliferation and tumor growth. Moreover, p68 induced NF-κB p50 accumulation in the nucleus through release of NF-κB p50 from IκBα and increased NF-κB p50 target luciferase transcription activity. Knockdown of NF-κB p50 rescued the phenotypes induced by p68 both in vitro and in vivo. We concluded that p68 induces glioma tumor growth through binding with NF-κB p50, regulating NF-κB p50 nucleus accumulation and transcription activity.

Regulation of sarco(endo)plasmic reticulum Ca2+-ATPase and calsequestrin gene expression in the heart

The precise control of Ca(2+) levels during the contraction-relaxation cycle in cardiac myocytes is extremely important for normal beat-to-beat contractile activity. The sarcoplasmic reticulum (SR) plays a key role controlling calcium concentration in the cytosol. The SR Ca(2+)-ATPase (SERCA2) transports Ca(2+) inside the SR lumen during relaxation of the cardiac myocyte. Calsequestrin (Casq2) is the main protein in the SR lumen, functioning as a Ca(2+) buffer and participating in Ca(2+) release by interacting with the ryanodine receptor 2 (RyR2) Ca(2+)-release channel. Alterations in normal Ca(2+) handling significantly contribute to the contractile dysfunction observed in cardiac hypertrophy and in heart failure. Transcriptional regulation of the SERCA2 gene has been extensively studied and some of the mechanisms regulating its expression have been elucidated. Overexpression of Sp1 factor in cardiac hypertrophy downregulates SERCA2 gene expression and increased levels of thyroid hormone up-regulates its transcription. Other hormones such norepinephrine, angiotensin II, endothelin-1, parathyroid hormone, prostaglandin-F2α, as well the cytokines tumor necrosis factor-α and interleukin-6 also downregulate SERCA2 expression. Calcium acting through the calcineurin-NFAT (nuclear factor of activated T cells) pathway has been suggested to regulate SERCA2 and CASQ2 gene expression. This review focuses on the current knowledge regarding transcriptional regulation of SERCA2 and CASQ2 genes in the normal and pathologic heart.

Ascorbic Acid Uptaken by Sodium-Dependent Vitamin C Transporter 2 Induces βhCG Expression through Sp1 and TFAP2A Transcription Factors in Human Choriocarcinoma Cells

Vitamin C [ascorbic acid (AA)] is transported by sodium-dependent vitamin C transporters (SVCT) 1 and 2, and our previous studies show AA induces a dramatic production of steroid hormones in human choriocarcinoma cells. However, whether AA induces the production of placental polypeptide hormones remains unknown. Here we investigated the mechanisms governing AA-induced β-human chorionic gonadotropin (hCG) expression. Frozen sections from human term placentas were used for immunostaining of SVCT, and βhCG mRNA expression and its production in primary human placental cytotrophoblasts and JEG-3 cells were examined by quantitative RT-PCR and ELISA, respectively. Knockdown of SVCT2, transcription factor activating enhancer-binding protein 2α (TFAP2A), or specificity protein-1 (Sp1) expression was achieved by retrovirus-mediated short hairpin RNA, and the transcriptional factors responsible for AA-induced βhCG expression was identified by reporter constructs. Both SVCT1 and SVCT2 are expressed in human term placentas. SVCT2 is predominantly localized in the syncytial layer, whereas SVCT1 is predominantly distributed in the villous core. AA dramatically induces βhCG mRNA expression and its production in JEG-3 cells and primary human cytotrophoblasts, and knockdown of SVCT2 expression in JEG-3 cells significantly decreases AA-induced βhCG expression. Data from βhCG5 construct and its deletion mutants further indicate that AA induces βhCG5 transactivation through Sp1 and TFAP2A transcriptional factors, and silence of Sp1 and/or TFAP2A expression significantly decreased AA-induced βhCG5 reporter activity and βhCG expression as well. The present study revealed the novel effects of AA on polypeptide hormone, βhCG, production and the potential mechanisms governing AA-induced βhCG expression, suggesting the potentially indispensable roles of AA in placental endocrine and pregnant maintenance.

Genome-wide localization and expression profiling establish Sp2 as a sequence-specific transcription factor regulating vitally important genes

The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes.

Sulforaphane Induction of p21Cip1 Cyclin-dependent Kinase Inhibitor Expression Requires p53 and Sp1 Transcription Factors and Is p53-dependent

Sulforaphane (SFN) is an important cancer preventive agent derived from cruciferous vegetables. We show that SFN treatment suppresses normal human keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). SFN treatment produces a concentration-dependent increase in p21(Cip1) promoter activity via a mechanism that involves stabilization of the p53 protein leading to increased p53 binding to the p21(Cip1) promoter p53 response elements. The proximal p21(Cip1) promoter GC-rich Sp1 factor binding elements are also required, as the SFN-dependent increase is lost when these sites are mutated. SFN treatment increases Sp1 binding to these elements, and the response is enhanced in the presence of exogenous Sp1 and reduced in the presence of ΔN-Sp3. CpG island methylation alters p21(Cip1) promoter activity some systems; however, expression in SFN-treated keratinocytes does not involve changes in proximal promoter methylation. The promoter is minimally methylated, and the methylation level is not altered by SFN treatment. This study indicates that SFN increases p21(Cip1) promoter transcription via a mechanism that involves SFN-dependent stabilization of p53 and increased p53 and Sp1 binding to their respective response elements in the p21(Cip1) promoter. These results are in marked contrast to the mechanisms observed in skin cancer cell lines and suggest that SFN may protect normal keratinocytes from damage while causing cancer cells to undergo apoptosis.

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 5291: A novel gain of function of deltaNp63 in tumor angiogenesis

Abstract Background: The p53 families of transcription factors (p53, p63, and p73) are known to be involved in cell stress response, development and tumor suppression. It has been shown that multiple splicing sites and alternative promoter regions lead to each of these proteins having multiple isoforms. The TA isoforms, which contain the transactivation domain, have been shown to induce apoptosis and inhibit cell-cycle progression, suppressing tumorigenesis, while the deltaN isoforms enhance proliferation and inhibit apoptosis, promoting tumorigenesis. Previous studies have shown that the deltaN isoforms are overexpressed in various types of tumors and somehow modulate the expression of proangiogenic and antiangiogenic factors in a different way thus determining the overall angiogenic activity of tumors. Methods: Since expression of deltaNp63 isoform in primary tumors has been shown to correlate with poor prognosis, we attempted to analyze their relationships with tumor angiogenesis. One quick assessment of angiogenesis is the measurement of the ability of endothelial cells to form three-dimensional structures (tube formation) that mimics angiogenesis in vitro. In the presence of angiogenic mediators, endothelial cells form tubes on collagen or fibrin clot coated dishes. To test whether overexpression of oncogenic deltaNp63 in HEK-293T can stimulate tube formation in endothelial cells (like HUVEC), we performed the endothelial tuber formation assay. In addition, we tested in human primary human cells whether there is any a strong correlation between overexpression of oncogenic deltaNp63 isoform and VEGF secretion. Moreover, we performed gene affymetrix as well protein micro array analysis in primary cells compared to cells overexpressed deltaNp63 isoform. In addition, we used siRNA technics to verify any potential role of deltaNp63 isoform in tumor angiogenesis Results: We found that overexpression of oncogenic isoform of p53 family member deltaNp63 in HEK-293T cells resulted in stimulation of tubular structures in HUVEC cells, which can be observed and counted under the microscope. As 293T cells lack functional p53, these results strongly suggest a gain of function for deltaNp63 isoform. In addition, in contrast to pcDNA-electroporated NHFB, used as a control for detection of endogenous VEGF, deltaNp63 overexpressed NHFB show 4 to 5-fold increased VEGF production. Interestingly, our gene expression array showed that ADAMTS8 were induced around 20-Fold in the presence of oncogenic ΔNp63. Conclusions: We found that deltaNp63beta increased VEGF and ADAMTS8 production by p53 independent manner. Our further studies showed that elevated deltaNp63 induced stabilization of HIF-1alpha protein resulting in VEGF secretion in p53 deficient cells. Furthermore, we showed, in the presence of deltaNp63beta isoform, the stabilization of HIF-1alpha and induced ADAMTS8 secretion was dependent on increased STAT3 phosphorylation. 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 5291. doi:1538-7445.AM2012-5291

Abstract 282: Knockdown of N-Myc and concurrent treatment with apigenin controlled growth of human malignant neuroblastoma cells having N-Myc amplification

Abstract Malignant neuroblastomas, which mostly occur in children, are frequently associated with N-Myc amplification and poor prognosis. Conventional chemotherapeutic agents are not very effective for controlling malignant neuroblastomas having N-Myc amplification. Notably, N-Myc amplification has profound implication in sustained growth of malignant neuroblastomas because N-Myc amplification is associated with inhibition of differentiation and apoptosis and promotion of angiogenesis and invasion. We have hypothesized that knockdown of N-Myc using short hairpin RNA (shRNA) plasmid may increase anti-cancer efficacy of a flavonoid such as apigenin (APG) in human malignant neuroblastoma cells that are known to harbor N-Myc amplification. Our confocal laser scanning immunofluorescence microscopy following staining of the cells with FITC conjugated N-Myc antibody and DAPI nuclear stain showed that N-Myc was highly expressed in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines, moderately in IMR32 cell line, and slightly in SH-SY5Y cell line. Different levels of N-Myc expression in these cell lines were also confirmed by flow cytometric analyses. We selected SK-N-DZ and SK-N-BE2 cell lines, which showed high N-Myc expression, for further studies. We used semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting to determine the levels of knockdown of N-Myc in SK-N-DZ and SK-N-BE2 cell lines following treatments with nothing (control), scrambled shRNA, N-Myc shRNA, APG, and N-Myc shRNA plus APG. Combination therapy caused the highest levels of knockdown of N-Myc in both cell lines. Knockdown of N-Myc induced neuronal differentiation with changes in morphological features (increased cell length and neurite growth) and biochemical features including increased expression of neurofilament protein (NFL), neuron specific enolase (NSE), and e-cadherin and decreased expression of Notch-1, Id2, catalytic subunit of human telomerase reverse transcriptase (hTERT), and proliferating cell nuclear antigen (PCNA). Our in situ Wright staining and Annexin V-FITC/PI staining showed that combination therapy was highly effective in inducing morphological and biochemical features of apoptosis. Combination therapy caused activation of caspase-8, cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, activation of calpain and caspase-3, and proteolysis of α-spectrin and ICAD. Combination therapy markedly decreased the migration of cells through matrigel and down regulated N-Myc driven survival factors (phospho-Akt and p65 NF-κB), angiogenic factors (VEGF and b-FGF), and invasive pathways (MMP-2 and MMP-9) in malignant neuroblastoma cells. Collectively, knockdown of N-Myc and concurrent APG treatment can be a promising therapeutic strategy for controlling growth of human malignant neuroblastoma cells. 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 282. doi:1538-7445.AM2012-282

Abstract 435: Targeted exome sequencing to understand tumor progression and identify targeted therapies

Abstract There is a large effort in the public domain to systematically perform DNA and RNA sequencing on large numbers of tumor samples. These efforts will bring us to a greater understanding of tumor biology and lead to identification of new tumor drivers. However, these efforts may fall short in allowing us to understand the progression of tumor resistance, relapse and metastasis, factors which make tumors difficult to treat and increase mortality rates. We are currently performing targeted DNA sequencing on 4000 tumor samples. We have selected 1321 genes, 5 GB of the genome to sequence. Genes were selected through a thorough review of the literature, mutation databases and key pathways in tumorigenesis such as growth factor signaling, DNA damage, p53 signaling, cell cycle and apoptosis. Our effort not only includes a diverse panel of breast, colon, ovarian, and kidney tumors but we have also sequenced pancreatic, liver, esophageal, cervix, endometrial, melanoma, CLL, DLBCL and carcinoid tumors. Our dataset also includes approximately 300 matched tumor-metastatic pairs and an additional 550 metastatic samples. Clinical phenotypes, treatment information, gene expression profiling and copy number variation data is available for all samples. To achieve the next level of care for cancer patients we must understand the biology of the tumors we are trying to treat, which are often metastatic and standard of care resistant. We believe that increasing our efforts in targeted DNA sequencing of key tumor and metastatic samples will allow us to achieve these goals. 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 435. doi:1538-7445.AM2012-435

Abstract 5025: Regulation of miR-145 expression by Foxo3a, C/EBP-β and p53 in cancer cells

Abstract Background: MicroRNAs are master gene regulators that control gene expression through the posttranscriptional repression mechanism. Accumulating evidence indicates that microRNAs are often dysregulated in human cancers, and they may function as oncogenes or tumor suppressors. Our laboratory and other groups have previously shown that miR-145 is a tumor suppressor that is downregulated in a variety of tumors. However, it is not fully understood about the underlying mechanism of miR-145 regulation. In this study, we characterized miR-145 regulation involving transcription factors FoxO3a, C/EBP-β and p53 in cancer cells. Methods: We previously constructed the miR-145 promoter luciferase reporter in pGL3-basic, which was used in this study to determine the effect of FoxO3a, C/EBP-β or p53 on miR-145 promoter activity. p53 expression vector was constructed in pEGFP; C/EBP-β and FoxO3a were cloned into pCDH lentiviral vector carrying copGFP. Western blot and fluorescence microscope were used to determine expression of exogenous genes. Endogenous miR-145 levels were determined by TaqMan real time PCR after reverse transcription as well as by in situ hybridization. The expression vectors or luciferase reporter were introduced into cells by either transfection or infection. Results: We showed that miR-145 functions a downstream effector of Akt, and impacts tumor growth and invasion. For example, suppression of Akt by serum starvation or PI3K inhibitor LY29 significantly activates p53, which in turn binds to the miR-145 promoter and induces miR-145 expression. Furthermore, we showed that in addition to p53, FoxO3a also functions a positive regulator of miR-145. Of considerable interest, FoxO3a induces miR-145 expression in the mutant p53 background; however, in the wild type p53 background, p53 suppresses the ability of FoxO3a to induce miR-145. On the other hand, C/EBP-β serves as a negative regulator of miR-145 in both wild type p53 and mutant p53 backgrounds, which is likely in part through regulation of Akt activity. Experiments are underway to further dissect the regulation of miR-145. Conclusion: Together, these results suggest a sophisticated regulatory system controlling miR-145 expression in cancer cells. Thus, a better understanding of this regulatory network will aid in the identification of novel cancer biomarkers and therapeutic targets, and improve cancer diagnosis and treatment. 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 5025. doi:1538-7445.AM2012-5025

Abstract 2345: Amplification of mouse chromosome 4 in chemically induced and invasive mouse lung adenocarcinoma

Abstract Lung cancer is the leading cause of cancer death in the United States and is ranked second only to bladder cancer in proportion of cases due to past occupational exposures. Though the genetic changes associated with lung cancer are not well understood, the pattern of mutations observed in lung adenocarcinoma from tobacco exposed patients is distinct from that of lung adenocarcinoma from unexposed patients. We used Spectral Karyotyping (SKY), mapping with fluorescently labeled genomic clones (FISH), comparative genomic hybridization (CGH), expression array, real time polymerase chain reaction and Western blot to analyze 15 primary adenocarcinoma and 9 pairs of high and low invasive cell cultures to detect molecular changes. The medial portion of chromosome 4 was deleted in 67% of all of the cell strains. Duplication of the proximal region of chromosome 4 occurred in 22% of the spontaneously-occurring high-invasive cells strains and 83% of the chemically-induced high-invasive cell cultures. Mouse chromosome 1 and 15 were amplified in 90% of the high-invasive cell strains. FISH mapping further narrowed the region of deletion of chromosome 4 to 39.6 centimorgans (cM) and the region of duplication to 10-35 cM. Expression array and real time PCR analysis demonstrated increased expression of the cell cycle inhibitory factor p16 and the apoptotic factor Cathepsin D in all of the tumor cell strains. The expression of cyclooxygenase 2 (COX-2), Kruppel like factor-4 (KLF4), Cyclin E and c-myc was significantly higher in the high-invasive cells strains compared to the low-invasive cell strains. Western blotting showed increased Cox-2, Cyclin E, KLF4 and c-myc proteins and decreased expression of Cathepsin D. In addition, the KLF4 protein was higher in the chemically-induced cell strains compared to the spontaneously-occurring cell strains while COX-2 was higher in the spontaneously-occurring cell strains. The KLF4 protein was identified in the tumor cell strains at the expected size of 55 KD as well as a smaller 45 KD form. Coordinated expression of KLF4 and c-myc is important in the induction of a stem cell phenotype and may be important in chemical carcinogenesis. The higher COX-2 in the spontaneous tumors indicates the importance of inflammation in spontaneous tumor induction. The homologous linkage groups on human chromosomes 9p21, 1p36, 9q and 8q are altered in asbestos-induced human lung adenocarcinoma. Alteration in copy number and expression of these genes may play a functional role in lung cancer development. 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 2345. doi:1538-7445.AM2012-2345

Transcriptional regulation mechanisms of hypoxia-induced neuroglobin gene expression

Ngb (neuroglobin) has been identified as a novel endogenous neuroprotectant. However, little is known about the regulatory mechanisms of Ngb expression, especially under conditions of hypoxia. In the present study, we located the core proximal promoter of the mouse Ngb gene to a 554bp segment, which harbours putative conserved NF-κB (nuclear factor κB)- and Egr1 (early growth-response factor 1) -binding sites. Overexpression and knockdown of transcription factors p65, p50, Egr1 or Sp1 (specificity protein 1) increased and decreased Ngb expression respectively. Experimental assessments with transfections of mutational Ngb gene promoter constructs, as well as EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays, demonstrated that NF-κB family members (p65, p50 and cRel), Egr1 and Sp1 bound in vitro and in vivo to the proximal promoter region of the Ngb gene. Moreover, a κB3 site was found as a pivotal cis-element responsible for hypoxia-induced Ngb promoter activity. NF-κB (p65) and Sp1 were also responsible for hypoxia-induced up-regulation of Ngb expression. Although there are no conserved HREs (hypoxia-response elements) in the promoter of the mouse Ngb gene, the results of the present study suggest that HIF-1α (hypoxia-inducible factor-1α) is also involved in hypoxia-induced Ngb up-regulation. In conclusion, we have identified that NF-κB, Egr1 and Sp1 played important roles in the regulation of basal Ngb expression via specific interactions with the mouse Ngb promoter. NF-κB, Sp1 and HIF-1α contributed to the up-regulation of mouse Ngb gene expression under hypoxic conditions.

Expression and clinical significance of transcription factor Sp1 and vascular endothelial cell growth factor in renal cell carcinoma

Objective To investigate the expression and clinical significance of transcription factor Spl and vascular endothelial cell growth factor （VEGF） in advanced renal cell carcinoma （RCC）. Methods The immunohistochemical PV-9000 method was used to study the expressed level of Spl and VEGF protein in 73 cases of advanced RCC. Results The positive rate of Spl and VEGF protein in advanced RCC was 75.3% and 83.6% respectively, the positive expression of Spl and VEGF showed a directly cor- relation. Conclusion Spl and VEGF may play an important role in the carcinogenesis and progression of RCC, the symphysial detection of the two proteins has a definite value in evaluating the prognosis of RCC. Key words: Renal cell carcinoma; Transcription factor Spl; Vascular endothelial cell growth factor; Positive expression

Elevated levels of hypoxia‐inducible microRNA‐210 in pre‐eclampsia: new insights into molecular mechanisms for the disease

Pre-eclampsia is a leading cause of maternal and foetal morbidity and mortality worldwide. Insufficient uteroplacental oxygenation is believed to be responsible for the disease. However, what molecular events involve in hypoxic responses and how they affect placental development remain unclear. Recently, miRNAs have emerged as a new class of molecules in response to hypoxia. We show here that the expression of microRNA-210 (mir-210) is up-regulated in patients with pre-eclampsia, as well as in trophoblast cells cultured under hypoxic conditions. Ectopic expression of mir-210 inhibited the migration and invasion capability of trophoblast cells. Ephrin-A3 and Homeobox-A9, which related with cell migration and vascular remodelling, were then experimentally validated as the functional targets of mir-210 both in vivo and in vitro. Using luciferase reporter, chromatin immunoprecipitation (ChIP) and small interfering RNA (siRNA) experiments, we finally identified a new transcriptional mechanism that the overexpression of mir-210 under hypoxia was regulated by NF-κB transcriptional factor p50, apart from the well-known HIF 1α. Taken together, our study implicates an important role for mir-210 in the molecular mechanism of pre-eclampsia.