Cytoplasmic Accumulation of the RNA-binding Protein HuR Stabilizes the Ornithine Decarboxylase Transcript in a Murine Nonmelanoma Skin Cancer Model

Abstract

Ornithine decarboxylase (ODC) is the first and usually ratelimiting enzyme in the polyamine biosynthetic pathway. Under normal physiological conditions, polyamine content and ODC enzyme activity are highly regulated. However, the induction of ODC activity is an early step in neoplastic transformation. The studies described here use normal mouse keratinocytes (C5N cells), and spindle carcinoma cells (A5 cells) to explore the regulation of ODC in nonmelanoma skin cancer development. Previous results have shown that induction of ODC activity is both necessary and sufficient for the promotion of skin tumors. We see a marked increase in ODC enzyme activity in A5 cells compared with C5N keratinocytes, which correlates with a 4-fold stabilization of ODC mRNA. These data suggest that ODC is post-transcriptionally regulated in skin tumor development. Thus, we sought to investigate whether the ODC transcript interacts with the RNA-binding protein HuR, which is known to bind to and stabilize its target mRNAs. We show that HuR is able to bind to the ODC 3-UTR in A5 cells but not in C5N cells. Immunofluorescence results reveal that HuR is present in both the nucleus and cytoplasm of A5 cells, whereas C5N cells exhibit strictly nuclear localization of HuR. Knockdown experiments in A5 cells showed that when HuR is depleted, ODC RNA becomes less stable, and ODC enzyme activity decreases. Together, these data support the hypothesis that HuR plays a causative role in ODC up-regulation during nonmelanoma skin cancer development by binding to and stabilizing the ODC transcript. Ornithine decarboxylase (ODC)2 catalyzes the conversion of the amino acid ornithine to the diamine putrescine, which is subsequently converted to the higher polyamines spermidine and spermine (1, 2). Polyamines are small, ubiquitous polycations and are necessary for normal cell growth and development. Thus, the ablation of ODC is lethal in utero (3). Because of their charge, polyamines are able to bind to RNA, DNA, and proteins and thereby influence gene expression (4). Both polyamine content and ODC enzyme activity are tightly regulated in cells, and ODC is induced in response to a variety of proliferative stimuli by alterations in its transcription, translation, and protein degradation (5-10). A link between neoplastic transformation and increased ODC enzyme activity, as well as increases in intracellular putrescine and spermidine, have been well documented in animal models of skin carcinogenesis and other epithelial tumors (11-17). Moreover, a link between human nonmelanoma skin cancer (NMSC), the most prevalent cancer in the United States, and elevated ODC enzyme activity levels has been described (18). The induction of ODC activity during tumor development has been attributed to both increased Odc gene transcription and translation of the ODC mRNA (9, 19).Interestingly, changes in intracellular polyamine pools have also been shown to affect the localization of RNA-binding proteins (RBPs), which influence the stability of their target mRNA transcripts (2...

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

“…We have shown previously using keratinocyte-derived cell lines that the RBP HuR associates with the ODC 3′UTR and stabilizes the ODC message in carcinoma cells but not in non-transformed cells [17]. To confirm that those results are not limited to the skin, we first performed an experiment designed to determine the intracellular localization of HuR in unstimulated parental and Ras12V-transformed RIE-1 cells (Figure 5A).…”

“…We have recently shown that the ODC transcript is stabilized by the RBP HuR in cells derived from skin carcinomas [17]. To determine whether changes in rate of mRNA decay could contribute to the dramatic ODC activity induction seen in Ras12V cells, we examined ODC RNA stability in both cell lines after exposure to Actinomycin D. The t 1/2 for RIE-1 ODC RNA was 3.0 h (Figure 1B, C), while the t 1/2 in Ras12V cells was calculated to be 23.7 h, indicating an 8-fold increase in stability (Figure 1B, 1C).…”

“…Western blot analysis was used to examine the association of HuR with the pull-down materials. For analysis of HuR binding to the DEL02 truncation mutant, biotinylated probes were synthesized and analyzed as described previously [17]. Mutation of the AUUUA sequence to GGGUA was by site-directed mutagenesis using the Quikchange Mutagenesis Kit as per the manufacturer’s instructions (Stratagene, La Jolla, CA).…”

“…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 ELAV protein family HuR associates with ODC mRNA in transformed cells and causes the ODC transcript to be stabilized [17]. Our results described here suggest that changes in ODC mRNA stability are mediated by cis -acting elements within the ODC 3′UTR and pathways downstream of mTORC1 play a role in this regulation at least in part by controlling the association of HuR with the ODC transcript.…”

Keratin-6 driven ODC expression to hair follicle keratinocytes enhances stemness and tumorigenesis by negatively regulating Notch

“…However, its expression and activity are induced in response to various external stimuli including tumor promoting agents [4, 5]. ODC is over-expressed in human non-melanoma skin cancers (NMSCs), including squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs), which are the most common human neoplasm [6]. Solar ultraviolet B (UVB), the major etiologic factor for NMSCs in humans induces ODC in the skin.…”