Vascular endothelial growth factor receptor-2 expression is induced by 17beta-estradiol in ZR-75 breast cancer cells by estrogen receptor alpha/Sp proteins

Vascular endothelial growth factor receptor-2 kinase insert domain receptor (VEGFR2/KDR) is critical for angiogenesis, and VEGFR2 mRNA and protein are expressed in ZR-75 breast cancer cells and induced by 17beta-estradiol (E2). Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich region is required for both basal and hormone-induced transactivation, and mutation of one or both of the GC-rich motifs at -58 and -44 results in loss of transactivation. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that Sp1, Sp3, and Sp4 proteins bind the GC-rich region of the VEGFR2 promoter. Results of the chromatin immunoprecipitation assay also demonstrate that ERalpha is constitutively bound to the VEGFR2 promoter and that these interactions are not enhanced after treatment with E2, whereas ERalpha binding to the region of the pS2 promoter containing an estrogen-responsive element is enhanced by E2. RNA interference studies show that hormone-induced activation of the VEGFR2 promoter constructs requires Sp3 and Sp4 but not Sp1, demonstrating that hormonal activation of VEGFR2 involves a nonclassical mechanism in which ERalpha/Sp3 and ERalpha/Sp4 complexes activate GC-rich sites where Sp proteins but not ERalpha bind DNA. These results show for the first time that Sp3 and Sp4 cooperatively interact with ERalpha to activate VEGFR2 and are in contrast to previous results showing that several hormone-responsive genes are activated by ERalpha/Sp1 in breast cancer cell lines.     

Nucleoside diphosphate kinase Nm23-H1 regulates chromosomal stability by activating the GTPase dynamin during cytokinesis

Chromosomal instability and the subsequent genetic mutations are considered to be critical factors in the development of the majority of solid tumors. Here, we describe how the nucleoside diphosphate kinase Nm23-H1, a protein with a known link to cancer progression, regulates a critical step during cytokinesis. Nm23-H1 acts to provide a local source of GTP for the GTPase dynamin. Loss of Nm23-H1 in diploid cells leads to cytokinetic furrow regression, followed by cytokinesis failure and generation of tetraploid cells. Loss of dynamin phenocopies loss of Nm23-H1, and ectopic overexpression of WT dynamin complements the loss of Nm23-H1. In the absence of p53 signaling, the tetraploid cells resulting from loss of Nm23-H1 continue cycling and develop classic hallmarks of tumor cells. We thus provide evidence that the loss of Nm23-H1, an event suspected to promote metastasis, may additionally function at an earlier stage of tumor development to drive the acquisition of chromosomal instability.     

Transfection of nm23-H1 increased expression of beta-Catenin, E-Cadherin and TIMP-1 and decreased the expression of MMP-2, CD44v6 and VEGF and inhibited the metastatic potential of human non-small cell lung cancer cell line L9981

Nm23 is a metastasis suppressor gene. In this report, we transfected nm23-H1 cDNA into L9981, a human large cell lung cancer cell line with nm23 negative expression, and made a stable transfectant. L9981-nm23-H1 cells exhibited lower cells proliferation rate, more G0/G1 phase growth and an increase in apoptosis with a dramatic decreased in the tumor cells ability to metastasize. L9981-nm23-H1 cells also demonstrated a significantly reduced lymph node and pulmonary metastatic capacity in vivo when injected into the nude mice. Furthermore, we used DNA microarray analysis to explore the change in expression of the metastasis-related genes in L9981-nm23-H1 cells. We found that the expression of beta-Catenin, E-Cadherin and TIMP-1 were significantly increased while expression MMP-2, CD44v6, and VEGF was dramatically decreased in L9981-nm23-H1, as confirmed by RT-PCR and western blot. These results demonstrated that nm23-H1 can suppress the mobility and metastatic capacity of cancer cells and the molecular mechanism by which nm23-H1 suppresses tumor metastasis may be via increasing the expression of metastasis-related genes such as beta-Catenin, E-Cadherin and TIMP-1 and decreasing the expression of MMP-2, CD44V6 and VEGF.     

Mechanism of transcriptional regulation of LRP16 gene expression by 17-beta estradiol in MCF-7 human breast cancer cells

LRP16 gene expression is induced by 17-betaestradiol (E2) via estrogen receptor alpha (ERalpha) in MCF-7 human breast cancer cells. A previous study also demonstrated that ectopic expression of LRP16 gene promoted MCF-7 cell proliferation. To explore the mechanism of hormone-induced LRP16 gene expression, the LRP16 gene promoter region (-2600 to -24 bp upstream of the LRP16 gene translation starting site) was analyzed in the present study by using different 5'-truncated constructs, and a luciferase reporter. The 5'-flanking sequence of -676 to -24 bp (pGL3-S5) was found to be E2-responsive. After exchange of the fragment from -213 to -24 bp with the TK gene proximal promoter region in pGL3-S5, E2 still induced reporter gene activity in MCF-7 and HeLa cells. Sequence analysis showed that the pGL3-S6 (-676 to -214) sequence contains two motifs that may contribute to E2-induced transactivation; namely, an estrogen-responsive element (ERE) half-site/Sp1 at -246 to -227 bp and an E-box site at -225 to -219 bp. Further deletion and mutation analysis of these two motifs indicated that both the 1/2 ERE and Sp1 binding sites were required for E2 action, while E-box deletion did not affect the luciferase activity in MCF-7 and HeLa cells. The results of gel mobility shift and chromatin immunoprecipitation assays confirmed that both ERalphaand Sp1 were required for hormone-induced transactivation, which involved both ERalphaand Sp1 directly binding to DNA. Taken together, these findings suggest that ERalphaand Sp1 play a role in activation of the human LRP16 gene promoter.     

Nm23-H1 expression in intrahepatic or extrahepatic metastases of hepatocellular carcinoma

Abstract: Aims/Background: Decreased expression of nm23, a putative metastasis suppressor gene, has been reported to be related to either intrahepatic metastasis or a poor prognosis in hepatocellular carcinoma (HCC). The aim of this study was to elucidate the true role of nm23-H1 expression in both intrahepatic and distant metastases of HCC. Methods: Thirteen patients with single-nodule HCC, seven patients with HCC having satellite nodules and seven patients with HCCs having extrahepatic metastases were included in this study. The expression of nm23-H1 protein was immunohistochemically examined in both primary and metastatic nodules. Results: Ten of 13 single-nodule HCCs were found to overexpress nm23-H1 protein. All main tumors, having satellite nodules, were found to overexpress nm23-H1 protein, except for two HCCs, which only partially expressed nm23-H1 protein. Regarding the nm23-H1 expression in intrahepatic metastases, most nodules overexpressed the protein. The expression of nm23-H1 was found to be low in only one intrahepatic metastasis specimen, while its primary tumor was also found to show a low expression of nm23-H1 protein. Microscopic portal vein invasion was found in three of the five patients studied, and all cancer cells in portal invasion overexpressed nm23-H1 protein. Nm23-H1 protein was expressed in all distant metastatic tumors and the staining intensity of most metastatic nodules was similar to that of the primary tumors. Conclusions: Our study demonstrated that nm23-H1 expression did not always decrease but instead tended to increase at both intrahepatic and extrahepatic metastatic sites. Based on these findings, nm23-H1 expression is not considered to be a reliable indicator of either intrahepatic or distant metastasis in HCC.     

Transcriptional activation of transforming growth factor alpha by estradiol: requirement for both a GC-rich site and an estrogen response element half-site

17beta-Estradiol (E2) induces transforming growth factor alpha (TGFalpha) gene expression in MCF-7 cells and previous studies have identified a 53 bp (-252 to -200) sequence containing two imperfect estrogen responsive elements (EREs) that contribute to E2 responsiveness. Deletion analysis of the TGFalpha gene promoter in this study identified a second upstream region of the promoter (-623 to -549) that is also E2 responsive. This sequence contains three GC-rich sites and an imperfect ERE half-site, and the specific cis-elements and trans-acting factors were determined by promoter analysis in transient transfection experiments, gel mobility shift assays and in vitro DNA footprinting. The results are consistent with an estrogen receptor alpha (ERalpha)/Sp1 complex interacting with an Sp1(N)(30) ERE half-site ((1/2)) motif in which both ERalpha and Sp1 bind promoter DNA. The ER/Sp1-DNA complex is formed using nuclear extracts from MCF-7 cells but not with recombinant human ERalpha or Sp1 proteins, suggesting that other nuclear factor(s) are required for complex stabilization. The E2-responsive Sp1(N)(x)ERE(1/2) motif identified in the TGFalpha gene promoter has also been characterized in the cathepsin D and heat shock protein 27 gene promoters; however, in the latter two promoters the numbers of intervening nucleotides are 23 and 10 respectively.     

Activation of adenosine deaminase in MCF-7 cells through IGF-estrogen receptor alpha crosstalk

Adenosine deaminase (ADA) regulates cellular levels of adenosine and deoxyadenosine, and 17beta-estradiol (E(2)) induces ADA mRNA in MCF-7 human breast cancer cells. IGF-I also induces ADA gene expression in these cells, and induction of this response through IGF activation of estrogen receptor alpha (ERalpha) was further investigated. IGF and other polypeptide growth factors induce reporter gene expression in MCF-7 cells cotransfected with ERalpha expression plasmid and pADA211, a construct containing the -211 to +11 region of the ADA gene promoter which is required for high basal and E(2)-inducible activity. Deletion analysis of this promoter demonstrates that IGF activates ERalpha/Sp1 interactions with multiple GC-rich sites in the promoter and this response is abrogated in cells transfected with ERalpha containing mutations at Ser(118) or Ser(163). IGF induces both MAPK (mitogen-activated protein kinase) and PI3-K (phosphatidylinositol-3-kinase) phosphorylation cascades in MCF-7 cells; however, using a series of inhibitors and dominant negative constructs, our results show that induction of ADA by IGF activation of ERalpha/Sp1 is dependent on the MAPK signaling pathway.     

Transcriptional activation of heat shock protein 27 gene expression by 17beta-estradiol and modulation by antiestrogens and aryl hydrocarbon receptor agonists

Heat shock protein 27 (Hsp 27) is expressed in mammary tumors and may play a role in tumor growth and response to anti-neoplastic drug therapy. 17beta-Estradiol (E2) induces Hsp 27 mRNA levels in MCF-7 human breast cancer cells, and we have investigated the comparative inhibitory mechanisms using the aryl hydrocarbon receptor (AhR) agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the direct-acting antiestrogen ICI 164,384. TCDD inhibited E2-induced Hsp 27 gene expression and analysis of the Hsp 27 gene promoter showed that the inhibitory response was associated with AhR interactions with a pentanucleotide motif at -3 to +2 in the promoter that corresponded to the core sequence of a dioxin responsive element. In contrast, ICI 164,384 induced Hsp 27 gene expression and reporter gene activity in MCF-7 cells and this represents one of the few examples of the estrogen receptor-alpha (ERalpha) agonist activity of the 'pure' antiestrogen ICI 164,384.     

Estrogen receptor/Sp1 complexes are required for induction of cad gene expression by 17beta-estradiol in breast cancer cells

The cad gene is trifunctional and expresses carbamoylphosphate synthetase/aspartate carbamyltransferase/dihydroorotase, which are required for pyrimidine biosynthesis. Cad gene activities are induced in MCF-7 human breast cancer cells, and treatment of MCF-7 or ZR-75 cells with 10 nM 17beta-estradiol (E2) resulted in a 3- to 5-fold increase in cad mRNA levels in both cell lines. The mechanism of hormone-induced cad gene expression was further investigated using constructs containing the growth-responsive -90 to +115 (pCAD1) region of the cad gene promoter. E2 induced reporter gene (luciferase) activity in MCF-7 and ZR-75 cells transfected with pCAD1, which contains three upstream GC-rich and two downstream E-box motifs. Deletion and mutation analysis of the cad gene promoter demonstrated that only the GC boxes that bind Sp1 protein were required for E2 responsiveness. Results of electrophoretic mobility shift and chromatin immunoprecipitation assays show that both Sp1 and estrogen receptor alpha interact with the GC-rich region of the cad gene promoter. Moreover, in transactivation assays with pCAD1, hormone-induced transactivation was inhibited by cotransfection with dominant-negative Sp1 expression plasmid and small inhibitory RNA for Sp1, which silences Sp1 expression in the cells. These results demonstrate that, in common with many other genes involved in E2-induced cell proliferation, the cad gene is also regulated by a nonclassical ERalpha/Sp1-mediated pathway.     

Gene expression profiling of Nm23-H2 overexpressing CAL 27 cells using DNA microarray

Nm23-H1/NDPKA and Nm23-H2/NDPKB belong to a large family of NDP kinases, group of structurally and functionally closely related enzymes. The Nm23/NDPs are known to catalyse the transfer of terminal phosphates from ATP to other NTPs and dNTPs. Besides their role in the maintenance of the cells NTP pool the nm23 genes/proteins are known to have additional different biological functions, the most important being its metastasis suppressor activity. The complete picture of roles, actions and targets of nm23 genes/proteins is yet to be discovered. Our goal was to identify the downstream targets of Nm23-H2 by subjecting Nm23-H2 overexpressing CAL 27 cells (oral squamous cell carcinoma of the tongue) to microarray analysis. Using this powerful technology we identified genes, groups of genes and signalling pathways that could be clustered into several groups: apoptosis related genes, cell cycle and DNA damage, TGFbeta (transforming growth factor beta) signalling pathway and related molecules, WNT signalling pathway, differentiation and epithelial structural and related molecules, cell adhesion, metalloproteinases and their inhibitors, vesicular transport related molecules, proteasome associated, ubiquitin mediated proteolysis and several metabolic pathways. Based on these results we suggest that nm23-H2 might have an important role in oral squamous cell carcinoma which is to be confirmed by future studies. Key words: nm23-H2, DNA microarray, oral squamous cell carcinoma.     

Loss of heterozygosity of thenm23-H1 gene in human renal cell carcinomas

This study evaluates the potential contribution of thenm23-H1 gene to malignant transformation in patients with renal cell carcinoma. Using specific oligonucleotide primers for thenm23-H1 microsatellite repetitive sequence, gene instability was followed by polymerase chain reaction/loss of heterozygosity assay on 54 tumor specimens and the corresponding normal tissue samples. We also determined, immunohistochemically, the relative concentration and localization of thenm23-H1 protein product. From 77.7% informative cases, DNA from 6 tumors exhibited loss of heterozygosity, regardless of the tumor stage (TNM). Out of 39 samples analyzed, 30 were negative for Nm23-H1 protein, while the others were only slightly positive. No correlation with tumor stage was found. Normal renal tissue was also negative for this protein. Our results provide the evidence for loss of heterozygosity, followed by means of microsatellite tandem-repeat polymorphism, at thenm23-H1 locus in renal cell carcinoma. However, since no correlation was found between the tumor stage or metastatic potential on the one hand, and allelic loss and specific protein expression on the other, it seems thatnm23-H1 does not play a key role in the invasiveness of this tumor type.Abbreviations PCR polymerase chain reaction - LOH loss of heterozygosity