HORMONAL REGULATION OF CARBOXYPEPTIDASE-D GENE TRANSCRIPTION IN BREAST CANCER CELLS

Abstract

Carboxypeptidase-D (CPD), bound to the plasma membrane, cleaves C-terminal arginine from extracellular substrates. Arginine is converted to nitric oxide (NO), which can promote tumour progression. We have previously reported that 17?-estradiol (E2) and prolactin (PRL) upregulate CPD mRNA/protein levels to increase NO production for the survival of human breast cancer cells. Androgen also upregulates CPD expression to increase NO production and survival of prostate cancer cells. The human CPD gene promoter contains a consensus ?-interferon-activated sequence (GAS) and several putative androgen response elements (AREs) that could potentially bind PRL-activated transcription factor Stat5 and the ligand-bound androgen receptor (AR), respectively. This study investigated regulation of the CPD gene by E2, PRL, and synthetic androgen R1881, in human MCF-7 and T47D breast cancer cell lines. CPD mRNA and protein levels were elevated by E2, PRL, and R1881, in a time- and dose-dependent manner. Upregulation of CPD mRNA by PRL and R1881 was abolished by actinomycin-D, suggesting transcriptional regulation by these two hormones. E2 acts by increasing CPD mRNA stability. The 2.0-kbp CPD gene promoter construct, containing a consensus GAS and three putative AREs, was stimulated by PRL and R1881, but not E2. PRL- and R1881-stimulated CPD promoter activities were not affected by deletion of ARE-2 and ARE-3, suggesting that the GAS, and in particular, ARE1, are active hormone response elements. PRL-stimulated GAS-CPD promoter activity was abolished by the mutation of GAS ( GAS-CPD, ARE-1 intact). Surprisingly, R1881 was unable to stimulate the same promoter. However, GAS-CPD promoter activity was restored when PRL and R1881 were administered together, and further enhanced by ectopic transfection of Stat5, suggesting cooperativity between Stat5 and the AR. Furthermore, ChIP analysis confirmed that PRL-activated Stat5 and the liganded AR bound to GAS and ARE- 1, respectively. In summary, PRL and R1881, acting through Stat5 and AR act in concert to stimulate CPD gene transcription and expression. E2 stabilizes the CPD mRNA to maintain CPD mRNA/protein levels. Taken together, our results implicate the cooperative action of the AR and PRL receptor signalling pathways in breast cancer.