BORIS, a paralogue of the transcription factor, CTCF, is aberrantly expressed in breast tumours.

BORIS (for brother of the regulator of imprinted sites), a paralogue of the transcription factor, CTCF, is a novel member of the cancer-testis antigen family. The aims of the present study were as follows: (1) to investigate BORIS expression in breast cells and tumours using immunohistochemical staining, western and real-time RT-PCR analyses and (2) assess potential correlation between BORIS levels in tumours with clinical/pathological parameters. BORIS was detected in all 18 inspected breast cell lines, but not in a primary normal breast cell culture. In 70.7% (41 of 58 cases) BORIS was observed in breast tumours. High levels of BORIS correlated with high levels of progesterone receptor (PR) and oestrogen receptor (ER). The link between BORIS and PR/ER was further confirmed by the ability of BORIS to activate the promoters of the PR and ER genes in the reporter assays. Detection of BORIS in a high proportion of breast cancer patients implies potential practical applications of BORIS as a molecular biomarker of breast cancer. This may be important for diagnosis of the condition and for the therapeutic use of BORIS. The ability of BORIS to activate promoters of the RP and ER genes points towards possible involvement of BORIS in the establishment, progression and maintenance of breast tumours.

Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia.

Oncogenic transformation and hypoxia both induce glut1 mRNA. We studied the interaction between the ras oncogene and hypoxia in up-regulating glut1 mRNA levels using Rat1 fibroblasts transformed with H-ras (Rat1-ras). Transformation with H-ras led to a substantial increase in glut1 mRNA levels under normoxic conditions and additively increased glut1 mRNA levels in concert with hypoxia. Using a luciferase reporter construct containing 6 kilobase pairs of the glut1 promoter, we showed that this effect was mediated at the transcriptional level. Promoter activity was much higher in Rat1-ras cells than in Rat1 cells and could be down-regulated by cotransfection with a dominant negative Ras construct (RasN17). A 480-base pair (bp) cobalt/hypoxia-responsive fragment of the promoter containing a HIF-1 binding site showed significantly higher activity in Rat1-ras cells than in Rat1 cells, suggesting that Ras might mediate its effect through HIF-1 even under normoxic conditions. Consistent with this, Rat1-ras cells displayed higher levels of HIF1-alpha protein under normoxic conditions. In addition, a promoter construct containing a 4-bp mutation in the HIF1 binding site showed lower activity in Rat1-ras cells than a construct with an intact HIF1 binding site. The activity of the latter construct but not the former could be down-regulated by RasN17, supporting the importance of the HIF1 binding site in regulation by Ras. The phosphatidylinositol 3-kinase inhibitor LY29004 down-regulated glut1 promoter activity and mRNA levels under normoxia and also decreased HIF1alpha protein levels in these cells. Collectively these results indicate that H-Ras up-regulates the glut1 promoter, at least in part, by increasing HIF-1alpha protein levels leading to transactivation of promoter through the HIF-1 binding site.

Epidermal growth factor receptor transcriptionally up-regulates vascular endothelial growth factor expression in human glioblastoma cells via a pathway involving phosphatidylinositol 3'-kinase and distinct from that induced by hypoxia.

Glioblastomas are highly vascular malignant brain tumors that often overexpress vascular endothelial growth factor (VEGF). They also frequently overexpress epidermal growth factor receptor (EGFR) and contain regions of hypoxia, both conditions that can induce VEGF. We examined VEGF regulation in U87 MG human glioblastoma cells and in U87/T691 cells, a clonal derivative that contains a truncated erbB2/Neu receptor that interferes with EGFR signaling through the formation of nonfunctional heterodimeric receptor complexes. U87/T691 cells contained approximately one-half as much VEGF mRNA as did U87 MG cells under normoxic conditions (21% oxygen). Pharmacological inhibition of EGFR, Ras, or PI(3) kinase, but not MAP kinase, led to a significant decrease in VEGF mRNA levels in U87 MG cells. VEGF promoter activity in transient transfections was decreased by either pharmacological or genetic inhibition of EGFR, Ras, or phosphatidylinositol 3'-kinase [PI(3) kinase]. However, inhibition of PI(3) kinase or EGFR did not completely abolish induction of VEGF mRNA by hypoxia (0.2% oxygen). Likewise, VEGF mRNA expression was induced 3-fold by hypoxia in EGFR-inhibited U87/T691 cells, comparable with the fold induction seen in parental U87 MG cells, although the absolute level of message under hypoxia was higher in U87 MG cells. In transient transfections, a luciferase reporter construct containing a 1.2-kb fragment of the VEGF promoter, lacking the known hypoxic-responsive element (HRE), showed up-regulation after EGF stimulation to the same degree as the full-length, 1.5-kb VEGF promoter construct retaining the HRE. Furthermore, activity of the HRE-deleted, 1.2-kb promoter luciferase reporter was down-regulated by PI(3) kinase inhibition. Therefore, in glioblastoma cells, transcriptional regulation of the VEGF promoter by EGFR appears to involve Ras/PI(3) kinase and to be distinct from signals induced by hypoxia.