Transcriptional and post-transcriptional regulation of Sprouty1, a receptor tyrosine kinase inhibitor in prostate cancer

Prostate Cancer and Prostatic Diseases (2011) 14, 279–285 & 2011 Macmillan Publishers Limited All rights reserved 1365-7852/11 www.nature.com/pcan REVIEW Transcriptional and post-transcriptional regulation of Sprouty1, a receptor tyrosine kinase inhibitor in prostate cancer M Darimipourain1, S Wang1, M Ittmann2 and B Kwabi-Addo1 1 Howard University Cancer Center, Washington, DC, USA and 2Department of Pathology, Baylor College of Medicine, Houston, TX, USA Sprouty1 (Spry1) is a negative regulator of fibroblast growth factor signaling with a potential tumor suppressor function in prostate cancer (PCa). Spry1 is downregulated in human PCa, and Spry1 expression can markedly inhibit PCa proliferation in vitro. We have reported DNA methylation as a mechanism for controlling Spry1 expression. However, promoter methylation does not seem to explain gene silencing in all PCa cases studied to suggest other mechanisms of gene inactivation, such as alterations in trans-acting factors and/or post-transcriptional activity may be responsible for the decreased expression in those cases. Binding sites for Wilm’s tumor (WT1) transcription factors EGR1, EGR3 and WTE are highly conserved between the mouse and human Spry1 promoter regions, suggesting an evolutionary conserved mechanism(s) involving WT1 and EGR in Spry1 regulation. Spry1 mRNA contains multiple microRNA (miRNA) binding sites in its 30 UTR region suggesting post-transcriptional control. We demonstrate that Spry1 is a target for miR-21-mediated gene silencing. miRNA-based therapeutic approaches to treat cancer are emerging. Spry1 is highly regulated by miRNAs and could potentially be an excellent candidate for such approaches. Prostate Cancer and Prostatic Diseases (2011) 14, 279–285; doi:10.1038/pcan.2011.33; published online 9 August 2011 Keywords: Spry1; transcription; microRNAs Introduction Prostate cancer (PCa) is the second most common malignancy and the second leading cause of cancer deaths in men in the United States. There is abundant evidence to indicate that inappropriate activation of fibroblast growth factor (FGF) receptor signaling has a critical role in the initiation and progression of PCa (for review see Kwabi-Addo, Ozen and Ittmann1). Sprouty (Spry) was originally identified in Drosophila as a negative regulator of FGF signaling during tracheal development.2 Subsequent studies have shown Spry to be a general inhibitor of growth factor-induced receptor tyrosine kinase (RTK) signaling pathways involved in Drosophila development and organogenesis.3–5 While Drosophila has only one Spry gene, at least four Spry homologs (Spry1–4) have been found in humans and mice.2,6,7 Mammalian Spry inhibits growth factor-i nduced cell responses by inhibiting the RTK-dependent Ras/mitogen-activated protein kinase signaling pathway.8–10 Several mechanisms for Spry inhibition of the RTK/Ras/mitogen-activated protein kinase pathway have been proposed, including blocking the interaction of the Grb2/SOS complex with the docking protein, FRS23,8 or the inhibition of Raf.11–13 Another characteristic of the Spry inhibitors is their regulation by growth Correspondence: Dr B Kwabi-Addo, Howard University Cancer Center, 2041 Georgia Ave N.W, Washington, DC 20060, USA. E-mail: Received 12 April 2011; revised 20 June 2011; accepted 25 June 2011; published online 9 August 2011 factors in a negative feedback loop, that is, their expression is dependent on the signaling pathway that they regulate.14 Specifically, growth factors regulate both the level of Spry transcript6 and, in some systems, the recruitment of Spry proteins to the plasma membrane.15 However, unlike Spry1, Spry2 and Spry4, whose expression can be inhibited in response to the downregulation of FGF signaling with the FGF receptor inhibitor SU5402, Spry3 is not inhibited by SU5402 treatment, suggesting that Spry3 might be involved in the regulation of another signaling pathway.14,16 Indeed, recent reports indicate that Spry3 has a role in axonal branching in a brain-derived neurotrophin factor-dependent manner;14,16 however, there is very little information about the role of Spry3 in PCa. Given that Spry1, 2 and 4 proteins can inhibit FGF signaling, they can potentially decrease the biological activities of FGFs in PCa cells and inhibit their ability to promote cancer progression. We have previously shown by immunohistochemical and quantitative real-time PCR (RT-PCR) analysis that Spry1 and Spry4 are downregulated in a subset of PCas tissues when compared with normal prostate tissues.17,18 We have also demonstrated that transient and sustained increased expression of Spry1 markedly inhibits PCa cell proliferation 17 whereas the sustained increased expression of Spry4 inhibited PCa cell migration.18 McKie et al.19 have observed that Spry2 expression is reduced in clinical PCa tissues when compared with BPH. The accumulating data indicate distinct differences in the functional roles for Spry1 and Spry4 in PCa cell lines. Transcriptional and post-transcriptional regulation of Sprouty1 M Darimipourain et al 280 The decrease in Spry expression in the human PCa, despite elevated levels of FGF ligands and FGF receptors, implies a loss of an important growth regulatory mechanism in PCas that may potentiate the effects of increased FGF and FGF receptor expression in PCa tissues and may represent a novel mechanism that facilitates aberrant RTK signaling in prostate carcinogenesis. We have previously demonstrated that DNA methylation in the Spry1 promoter region is responsible for downregulating Spry1 expression in PCa.20 However, promoter methylation does not seem to explain gene silencing in all PCa cases studied. For instance, in some PCa cases, where we detected low DNA methylation in the cancer tissues compared with the normal tissues, we also observed low Spry1 expression to suggest that other mechanisms of gene inactivation such as alterations in trans-acting factors and/or post-transcriptional activity may be responsible for the decreased expression in those cases. The purpose of the present study is to investigate the relative contribution of trans-acting factors and posttranscriptional regulation of Spry1 in PCa. Our data support a potential role of mir-21 in post-transcriptional regulation of Spry1 in PCa cells. Materials and methods Cell Culture and treatment The human PCa cell lines, PC3, DU145 and LNCaP, and the immortalized normal prostate epithelial cell line pNT1A were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) unless otherwise stated. LNCaP cells were treated with 10 ng ml1 of lipopolysaccharide (Sigma-Aldrich, St Louis, MO, USA) for 24 h in complete medium without antibiotics. Treated and untreated cells were harvested and extracted RNA was used in quantitative RT-PCR with Cancer miRNA Array (SA BioSciences, Freder (...truncated)