Regulation of a promoter of the fibroblast growth factor 1 gene in prostate and breast cancer cells

https://doi.org/10.1016/S0960-0760(98)00051-XGet rights and content

Abstract

FGF-1 mRNA is expressed in the prostate cancer cell lines LNCaP and PC-3 and in the breast carcinoma cell line MDA-MB-231. Levels of FGF-1 mRNA have been shown to be up-regulated by serum, phorbol esters, and combinations of growth factors. It was shown that the major FGF-1 mRNA species expressed following serum stimulation in MDA-MB-231 cells is FGF-1.C. To better understand the potential role of FGF-1 in human prostate and breast cancer, we began an analysis of the cis- and trans-acting elements of one of its promoters required for the serum, PMA, and androgen regulation in breast and prostate cancer cell lines. We show that FGF-1.C steady-state mRNA levels are increased following serum or PMA stimulation of PC-3 cells. Further, we determine the FGF-1.C transcription start site in PC-3 cells. By sequence analysis, we show that consensus AP1, AP2, and Sp1 sites and ARE- and CRE-near consensus elements are present in the immediate 5′ region of the FGF-1.C transcription start site. Gel-shift assays show that oligonucleotides containing FGF-1.C AP1, AP2, or Sp1 sequences form specific DNA–protein complexes with nuclear extracts from PC-3 cells. To determine if these or other cis-acting sequences are responsible for the serum, androgen, or growth factor regulation of FGF-1 expression, fragments of the FGF-1.C promoter region were cloned upstream of the luciferase reporter gene. We show that FGF-1 synergizes with androgen to enhance FGF-1.C transcription in LNCaP cells. We further show that the DNA fragment containing sequence up to 1614 nucleotides upstream of the FGF-1.C transcription start site is sufficient for stimulating promoter activity following serum treatment of MDA-MB-231 cells. Thus, FGF-1.C promoter contains sequences that are important for androgen or serum stimulation in prostate and breast cancer cells.

Introduction

The human fibroblast growth factor 1 (FGF-1) is one member of a growing number of proteins that constitute the fibroblast growth factor family1, 2, 3, 4. FGF-1 plays a significant role in several biological processes including cell growth and development and malignant transformation. It is a mitogen for mesoderm- and neuroectoderm-derived cells[1]. When overexpressed in NIH/3T3 cells, FGF-1 causes transformation; when injected into nude mice, it induces tumor formation[5]. FGF-1 has been shown to be a potent angiogenic factor both in vivo and in vitro[6]and as such may play a role in tumor progression by promoting tumor vascularization[7]or in proliferative diabetic retinopathy[8]. FGF-1 was shown to be an inducer of mesoderm development in the Xenopus embryo[9]. Its expression in the smooth muscle cells of the vascular wall has been implicated in atherosclerosis[10].

FGF and FGF receptor (FGFR) gene expressions have been detected in normal, benign, and malignant tissues as well as tumor cell lines. Penaul-Llorca et al.[11]found FGF-1 gene expression in many breast tumor samples tested and concomitant high-level gene expression of FGFR1, 2 and 4 in many of these same samples. Smith et al.[12]found FGF-1 protein levels to be higher in malignant breast tissues compared with normal or benign tissues. By contrast, Bansal et al.[13]and Anandappa et al.[14]found that FGF-1 expression is lower in breast cancer tissues than in normal breast tissues. FGF-1 expression has been reported in various breast and prostate cancer cell lines. For example, FGF-1 mRNA expression has been found in the malignant human mammary epithelial cell lines MDA-MB-231, MCF-7 and BT-2015, 16and the prostate carcinoma cell lines PC-3[17]and LNCaP[18].

FGF-1 mRNA levels are up-regulated by a variety of mitogens including serum19, 20, 21, phorbol esters21, 22and combinations of growth factors[21]. Depending on the cell type, serum either stimulates FGF-1 mRNA expression as in vascular smooth muscle cells19, 21, or decreases its expression as in lens epithelial cells[23]. Steroid hormones appear to regulate the growth of hormone-dependent carcinoma cells indirectly through steroid-induced polypeptide growth factors24, 25. In LNCaP cells, for example, Harris et al.[18]found that the levels of FGF-1 mRNA increased following androgen stimulation.

The human FGF-1 gene spans more than 100 kb and contains three protein-coding exons and at least four 5′ untranslated exons. Multiple FGF-1 transcripts arise from alternative splicing17, 26, multiple promoter usage[27], and alternative transcriptional terminations[28]. Splicing of upstream exons −1A, −1B, −1C, or −1D to the first protein-coding exon results in FGF-1 transcripts 1.A, 1.B, 1.C, and 1.D that are expressed in a tissue- and cell-specific manner17, 19, 26. The biological significance of having alternative 5′ untranslated exons may include transcript stabilization[29]and compartmentalization[30], and translational regulation[31].

To better understand the potential role of FGF-1 in human prostate and breast cancer, we began an analysis of the cis- and trans-acting elements of one of its promoters required for the serum, PMA, and androgen regulation in breast and prostate cancer cell lines. We show that FGF-1.C mRNA expression is increased following serum or PMA treatment of PC-3 cells. Using luciferase reporter analysis, we show that the sequence from −1614 to the FGF-1.C start site is sufficient to stimulate promoter activity following FGF-1 plus androgen treatment of LNCaP cells or serum treatment of MDA-MB-231 cells.

Section snippets

Chemicals

The synthetic androgen, 17β-hydroxyl-17α-methyl-estra-4,9,11-trien-3-one (R1881), was obtained from Roussel-UCLAF, Romainvelle, France. Dihydrotestosterone (DHT), testosterone, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma (St. Louis, MO). Epidermal growth factor (EGF) and FGF-1 were purchased from Upstate Biotechnology (Lake Placid, NY).

Cell culture

PC-3 cells (ATCC, passage number 16) were grown in DMEM (Gibco-BRL) supplemented with nonessential amino acids, 10% fetal bovine serum

FGF-1.C mRNA is increased following serum or PMA treatment of PC-3 cells

Since FGF-1 mRNA levels were shown to be up-regulated by serum19, 20, 21and phorbol esters21, 22, we specifically wanted to know which FGF-1 prmoter(s) is activated under the same conditions. PC-3 cells were grown to 80% confluency and then serum starved for 72 h in medium containing 0.5% FBS. Serum-starved cells were then stimulated with either 10% FBS or PMA (100 ng/ml) for 6 h, the time point shown previously to result in the greatest FGF-1.C stimulation in saphenous vein smooth muscle cells[19]

Discussion

The induction of the FGF-1.C mRNA by serum in PC-3 cells and MDA-MB-231 cells as demonstrated in RNase protection analysis (Fig. 1) and transient transfection analysis (Fig. 4, Fig. 7) provide model systems to study the signal transduction pathways involved in the stimulation of the FGF-1.C promoter. In the 5′-flanking sequences of FGF-1.C are consensus or near consensus AP1, AP2, CRE, and Sp1 sites (Fig. 3). Using EMSA, we showed binding of protein from PC-3 nuclear extracts to AP1, AP2, and

Acknowledgements

We thank Yang Liu for her technical help. This work was supported by grants R01CA45611 and R01DK47506 from the National Institutes of Health. I.-M.C. was supported by a Research Career Development award (K04CA01369) and R.A.P. was supported by a National Research Service Award (T32CA09498) from the National Institutes of Health.

References (47)

  • M. Imagawa et al.

    Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP

    Cell

    (1987)
  • J.T. Kadonaga et al.

    Promoter-specific activation of RNA polymerase II transcription by Sp1

    Trends Biochem. Sci.

    (1986)
  • S.L. Mcknight et al.

    The distal transcription signals of the herpes virus tk gene share a common hexanucleotide control sequence

    Cell

    (1984)
  • W.H. Burgess et al.

    The heparin-binding (fibroblast) growth factor family of proteins

    Annu. Rev. Biochem.

    (1989)
  • A. Tanaka et al.

    Cloning and characterization of an androgen-induced growth factor essential for the androgen-dependent growth of mouse mammary carcinoma cells

    Proc. Natl. Acad. Sci. U.S.A.

    (1992)
  • M. Miyamoto et al.

    Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property

    Mol. Cell. Biol.

    (1993)
  • P. Bunnag et al.

    Transformed phenotype conferred to NIH/3T3 cells by ectopic expression of heparin-binding growth factor 1/acidic fibroblast growth factor

    In vitro Cell. Dev. Biol.

    (1991)
  • J. Folkman et al.

    Angiogenic factors

    Science

    (1987)
  • E.G. Nabel et al.

    Recombinant fibroblast growth factor-1 promotes intimal hyperplasia and angiogenesis in arteries in vivo

    Nature

    (1993)
  • D. Fredj-Reygrobellet et al.

    Acidic FGF and other growth factors in preretinal membranes from patients with diabetic retinopathy and proliferative vitreoretinopathy

    Ophthalmic Res.

    (1991)
  • J.M. Slack et al.

    Mesoderm induction in early Xenopus embryos by heparin-binding growth factors

    Nature

    (1987)
  • E. Brogi et al.

    Distinct patterns of expression of fibroblast growth factors and their receptors in human atheroma and nonatherosclerotic arteries. Association of acidic FGF with plaque microvessels and macrophages

    J. Clin. Invest.

    (1993)
  • F. Penault-Llorca et al.

    Expression of FGF and FGF receptor genes in human breast cancer

    Int. J. Cancer

    (1995)
  • Cited by (25)

    • FGF1 and FGF2 mutations in preeclampsia and related features

      2016, Placenta
      Citation Excerpt :

      Over-expression of FGF1 by glomerular endothelial and mesangial cells is a key regulator of glomerular microcirculation and systemic BP [12]. FGF1 gene is localized on chromosome 5q31.3–33.2 [22], and contains three protein-coding exons [23], and its transcription is controlled by at least four distinct promoters expressed in several cell types, including vascular smooth muscle cells [24–26]. It is tempting to speculate that -1385A/G polymorphism may function in controlling FGF-1.C promoter activity [16,18].

    • No association of the C > T polymorphism that is located 1385 upstream from initial code of fibroblast growth factor 1 gene with Alzheimer's disease in Chinese

      2010, Brain Research
      Citation Excerpt :

      Multiple FGF1 transcripts arise from alternate promoter usage and alternative spicing of different 5′-untranslated exons. Splicings of upstream exons A, B, C and D to the first coding exon result in FGF1 transcripts A, B, C and D that are expressed in a tissue- and cell-specific manner respectively (Chiu et al., 2001; Myers et al., 1993; Payson et al., 1998). To date, two groups reported that a polymorphism (− 1385 G/A, rs34011) was associated with Alzheimer's disease in Japanese and Chinese respectively (Yamagata et al., 2004; Yao et al., 2006).

    • Promoter polymorphism in fibroblast growth factor 1 gene increases risk of definite Alzheimer's disease

      2004, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      However, functional role of the haplotype in its pathophysiology remains unclear. As the FGF1 gene contains alternative 5′-untranslated exons, the transcription is controlled by at least four distinct promoters in a tissue-specific manner [18–20]. Payson et al. [19] have reported that the sequence from −1614 to the FGF1 start site is sufficient to stimulate promoter activity.

    • The small GTPases Ras, Rac, and Cdc42 transcriptionally regulate expression of human fibroblast growth factor 1

      2000, Journal of Biological Chemistry
      Citation Excerpt :

      The 1A reporter was constructed by placing the 1A promoter region from −826 to +77 upstream of the luciferase reporter gene (pGL2-Basic vector, Promega Corp, Madison, WI). The FGF1B and -1C luciferase constructs have previously been described (5, 8). The previously described FGF1Dminigene (10) was used as DNA template to polymerase chain reaction amplify the FGF1D promoter region −150 to +40, and including 9 bp of the exon 1 sequence.

    View all citing articles on Scopus
    View full text