A high priority for the detection and eradication of prostate cancer (Pca) is the elucidation of specific target genes which are involved in the initiation and progression of this disease. We believe that we have identified one such target, EGR-1, which is overexpressed in prostate cancer. EGR-1 is an immediate early growth response gene and a member of a family of zinc-finger transcription factors including EGR-2, EGR-3, EGR-4, EGR-β£, and WT-1.
We conducted a detailed study of EGR-1 expression in PCa samples. EGR-1 mRNA was quantified in 96 graded PCa samples using in situ hybridization with a specific [S 35 ]labeled cRNA EGR-1 probe. EGR-1 mRNA was found to be significantly higher in PCa samples with Gleason score 8-10 than in PCa samples with a lower grade. Thus, EGR-1 expression increases concomitantly with an increase in the index of malignancy. These results were further corroborated with immunocytochemistry, which indicated that EGR-1 may be expressed primarily in the basal epithelial cells. In addition, these studies revealed that EGR-1 is detected primarily in the cytoplasm of normal and benign tissue, whereas it is predominantly nuclear in the cancerous tissue.
To further corroborate the subcellular localization of EGR-1 in cancer vs. noncancerous prostate, we performed immunocytochemistry on two human prostate cell lines from benign (BPH-1) and cancerous (PC-3) origin. EGR-1 protein was primarily localized in the cytoplasm of BPH-1 cells and in the nuclei (mostly in the nucleoli) of PC-3 cells. Interestingly, in all dividing cells (either BPH-1 or PC-3 cells), EGR-1 was preferentially associated with the mitotic spindle. EGR-1 is present as a 75-kDa protein in the nuclei of PC-3 cells and as a larger 93-kDa form in the cytoplasm of these cells. In agreement with the immunofluorescence data, higher levels of EGR-1 were found in the nuclei vs. the cytoplasm of cells when compared by equal amounts of protein loads. In addition, the nuclear form appears as a doublet, suggesting that this form might be phosphorylated.
In order to examine the effect of calcium on EGR-1 expression, we used the well-differentiated PCa cell line, LNCaP. Cells were maintained in DCC-FBS medium for 24 hr and then treated with 10 -7 M thapsigargin. Expression of EGR-1 mRNA increased rapidly by 2-fold within 20 min of treatment with thapsigargin. These levels reached a maximum of 4-fold induction between 40-60 min of treatment. Levels remained elevated for 2 hr and then decreased after 4 hr of treatment. This thapsigargin-induced EGR-1 expression was blocked by staurosporine, suggesting a role of PKC in EGR-1 regulation in PCa cells. In addition, the calmodulin inhibitor, trifluoroperazin, also suppressed the thapsigargininducive effect. These data suggest that in addition to PKC, a calmodulin-mediated pathway is involved in the regulation of EGR-1 expression in PCa cells. Moreover, since thapsigargin induces apoptosis in LNCaP cells, EGR-1 may also be involved in this process in PCa cells.
These data suggest that EGR-1 has potential prognostic value for prostate cancer detection. Moreover, it may provide a unique target for potential gene therapy.