Ornithine decarboxylase mRNA is stabilized in an mTORC1-dependent manner in Ras-transformed cells.

NIH Public Access Author Manuscript Biochem J. Author manuscript; available in PMC 2013 February 15. Published in final edited form as: Biochem J. 2012 February 15; 442(1): 199–207. doi:10.1042/BJ20111464. Ornithine decarboxylase mRNA is stabilized in an mTORC1dependent manner in Ras-transformed cells $watermark-text Sofia Origanti*,1, Shannon L. Nowotarski*,2, Theresa D. Carr*, Suzanne Sass-Kuhn*, Lan Xiao†,‡, Jian-Ying Wang†,‡,§, and Lisa M. Shantz* *Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA 17033, USA †Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A ‡Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A §Baltimore Veterans Affairs Medical Center, Baltimore, MD 21201, U.S.A $watermark-text SYNOPSIS $watermark-text Upon ras activation, ornithine decarboxylase (ODC) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumors. We compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed rat intestinal epithelial (RIE-1) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC RNA was stabilized 8-fold. Treatment with the specific mTORC1 inhibitor rapamycin or siRNA knockdown of mTOR destabilized the ODC message, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3′UTR results in significant stabilization of the ODC mRNA, which contains several AUrich regions within its 3′UTR that may act as regulatory sequences. Analysis of ODC 3′UTR deletion constructs suggests that cis-acting elements between bases 1969 and 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumor development. Keywords ornithine decarboxylase; mRNA stability; translation initiation; mTOR; rapamycin; HuR Corresponding author: Lisa M. Shantz, Ph.D., Associate Professor, Department of Cellular and Molecular Physiology H166, Room C4731, The Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA, phone: 717-531-1562, fax: 717-531-7667, . 1Current address: Sofia Origanti, Ph.D., Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA 2Current address: Shannon L. Nowotarski, Ph.D., The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA AUTHOR CONTRIBUTIONS: Sofia Origanti performed the majority of the experiments and was involved in writing and editing the manuscript. Shannon Nowotarski, Theresa Carr, Suzanne Sass-Kuhn and Lian Xiao performed some of the experiments. Jian-Ying Wang provided reagents and critical reading of the manuscript. Lisa Shantz had the original idea, secured funding, and was involved in writing and editing the manuscript. Origanti et al. Page 2 INTRODUCTION $watermark-text Increased activity of ornithine decarboxylase (ODC), the first biosynthetic enzyme of the polyamine pathway, has been demonstrated in a variety of human malignancies, including prostate cancer, squamous cell carcinoma of the skin and colon cancer (reviewed in [1]). Intracellular levels of ODC are tightly regulated, and multiple means of ODC dysregulation can occur in response to oncogenic stimuli. We have used in vitro and in vivo models of Ras activation to establish that ODC activity is regulated by and necessary for Ras-dependent cellular transformation, as well as transformation brought about by the Ras effectors MEK and eIF4E [2–5]. Activation of ODC transcription and protein synthesis is dependent on pathways downstream of Raf/MEK/ERK and PI3K/mTOR in both fibroblast and epithelial models [3, 6]. The cooperation of pathways controlled by Raf and PI3K/mTOR is necessary for complete Ras transformation of several types of epithelial cells (reviewed in [7]). Since most solid tumors are epithelial in origin, understanding how ODC synthesis is controlled by these pathways is crucial in defining the role of ODC in maintaining a transformed phenotype. $watermark-text Cap-dependent translational regulation of ODC through its 5′-untranslated region (5′UTR) is well-established, and ODC activity and translation are induced in eIF4E-overexpressing fibroblasts (4E-P2 cells) [2, 8]. However, our studies in rat intestinal epithelial cells (RIE-1 cells) described here suggest an alternate post-transcriptional regulatory mechanism for ODC protein synthesis. In this system, ODC synthesis is regulated primarily by changes in the levels of ODC RNA associated with polysomes, rather than changes in translation initiation. The mechanism of this regulation is a marked stabilization of the ODC mRNA in Ras12V-transformed RIE-1 cells (Ras12V cells) compared to their nontransformed parental controls, which appears to be regulated at least in part by pathways downstream of mTOR Complex 1 (mTORC1). Although the primary function of mTORC1 is in controlling the availability of eIF4E for translation initiation (reviewed in [9]), several studies show that TOR inhibition results in RNA stabilization. In S. Cerevisiae inhibition of TORC1 using the specific inhibitor rapamycin induced destabilization of multiple mRNAs, suggesting that TORC1 functions also involve regulation of mRNA turnover [10, 11]. In mammalian systems, rapamycin treatment of mouse embryo fibroblasts increased the degradation of mRNAs corresponding to Cyclin D1 and c-Myc in an Akt-dependent manner [12], while treatment of breast cancer MDA-MB-231 cells with rapamycin resulted in destabilization of IL-8 mRNA [13]. $watermark-text Regulation of mRNA stability is recognized to play a pivotal role in controlling gene expression. Sequences defined as adenylate- and uridylate-rich elements (AREs), which are classified based on the number and context of the sequence 5′-AUUUA-3′, are present within the 3′UTRs of many proto-oncogene, transcription factor and cytokine mRNAs (reviewed in [14, 15]), and can act as determinants of mRNA stability. The mouse, rat and human ODC 3′UTR sequences, each of which is between 600–700 bases in length, have several potential AREs within approximately 300 bases the stop codon. A number of regulatory proteins are known to interact with ARE sequences. These proteins not only control transcript decay, but can also influence translational efficiency, or cause the bound RNA transcript to move to a processing body (P-body) for storage [16]. We have shown recently that the ubiquitous member of the E (...truncated)